Protease Inhibitor-Containing Compositions, Compositions Comprising Same, and Methods for Producing and Using Same

ABSTRACT

Provided herein are methods and compositions for oral administration of therapeutic proteins, improved protease inhibitor preparations, methods for producing same, and compositions comprising same.

Benefit is claimed to U.S. Provisional Application 61/632,868, filedFeb. 1, 2012, and to U.S. Provisional Application 61/634,753, filed Mar.6, 2012, which are incorporated herein by reference in their entirety.

FIELD

Provided herein are methods and compositions for oral administration oftherapeutic proteins, improved protease inhibitor preparations, methodsfor producing same, and compositions comprising same.

BACKGROUND

Protein/peptide-based drugs are typically susceptible to degradation inthe gastrointestinal tract and/or are not efficiently absorbed into thebloodstream from the small intestine in bioactive form. Orally deliveredformulations for protein-based drugs such as insulin are being developed(Ziv et al 1994; Nissan et al 2000, Kidron et al 2004, Eldor et al2010A, Eldor et al 2010B). One such oral insulin product is scheduled tobe tested in Phase II trials and is currently being reviewed for INDstatus.

Trypsin inhibitors derived from soybean (Glycine max) are readilyavailable and are considered to be safe for human consumption. Theyinclude SBTI (soybean trypsin inhibitor), which is composed of KTI(Kunitz Trypsin Inhibitor), which inhibits trypsin, and BBI (Bowman-Birkinhibitor), which inhibits trypsin and chymotrypsin. Such trypsininhibitors are available for example from Sigma-Aldrich, St. Louis, Mo.,USA. Methods for preparing BBI are described for example in U.S. Pat.No. 7,404,973.

SUMMARY

The present inventor has discovered that commercially available SBTIpreparations produced highly variable results when used inpharmaceutical compositions. It was postulated that the activities ofKTI and BBI should be individually optimized in order to improve theactivity of the pharmaceutical compositions. To this end, SBTI wasobtained from a commercial source as separate preparations of KTI andBBI. However, each of these preparations was found to be contaminatedwith the other activity. Compounding the issue, significant variabilitywas found in the BBI activity of the preparations, particularlylarge-scale preparations, as evidenced by variable ability to preventdegradation of proteins (e.g. insulin) by intestinal enzymes.

Accordingly, in accordance with certain embodiments described herein, animproved method for the purification of SBTI was developed, in whicheach product was prepared under its own specifications to high levels ofactivity, and levels of high molecular weight (MW)-contaminants wereminimized. In accordance with other embodiments, the process avoids theuse of PEG and a second chromatography step and is characterized byhigher yield. In accordance with still other embodiments, the product isparticularly suitable for use in pharmaceutical compositions, forexample compositions for oral administration of therapeutic proteins. Inaddition, in accordance with yet other embodiments, the completeseparation of the KTI and BBI activities allows more precise modulationof the anti-trypsin and anti-chymotrypsin activities of pharmaceuticalcompositions comprising a therapeutic protein and the BBI and/or KTI,allowing for more robust and/or more reproducible in vivo activity ofthe pharmaceutical compositions.

It was further discovered that emulsifiers were required to convenientlyprepare large-scale preparations of peptide/protein-containing drugs.However, it was necessary to empirically test whether addition ofparticular emulsifiers could effectively prevent precipitation in theoil-based preparations without affecting the oral efficacy of theformulations. Other described embodiments thus relate to the presence ofparticular emulsifiers or combinations of emulsifiers, together withimproved SBTI, in oil-based pharmaceutical compositions containingtherapeutic peptides and proteins.

The terms “protein” and “peptide” are used interchangeably herein.Neither term is intended to confer a limitation of the number of aminoacids present, except where a limitation is explicitly indicated.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are by way of illustrative example and are notmeant to be taken as limiting the claimed invention.

FIGS. 1(A) AND 1(B) Flow diagrams of SBTI purification. FIG. 1(A): SBTIintermediate production. FIG. 1(B): Downstream purification to produceBBI and KTI.

FIG. 2. Chromatogram of DEAE Sepharose™ column separation. Fractions of0.3 L were collected during DEAE column elution.

FIG. 3. SDS-PAGE analysis of purified BBI and KTI. Electrophoresis wasperformed, the gel was scanned, and bands were quantified using anImageScanner™ III in conjunction with ImageQuant™ TL (both from GeneralElectric). Samples of 1 milligram per milliliter (mg./ml.) were loadedon a 20% Phastgel™. Lane 1: Control (old SBTI preparation). Lane 2:Purified KTI, Band quantity=100%. Lane 3: Purified BBI, lower bandquantity=89.2%, upper band quantity=10.8%.

FIG. 4. Small-scale column chromatography run report using improvedprocedure. Squares, circles, and triangles denote conductivity plot, pH,and OD₂₈₀, respectively. Vertical axes: conductivity plot(milliSiemens), pH, and OD₂₈₀ (arbitrary units). Horizontal axis: columnvolume and fraction number.

FIG. 5(A-C) 20% SDS-PAGE of fractions from chromatography shown in FIG.4. FIG. 5(A): Left-to-right presentation of fractions 4-18 and pooledfractions 5-11. “C” denotes prior art trypsin inhibitor (Sigma-Aldrichcat. no. T9003). FIGS. 5B AND 5C: Right-to-left presentation offractions 6-15, including the wash+flowthrough (W+FT) and standard(Trypsin inhibitor from Glycine max (soybean); “ST”).

FIG. 6. SDS-PAGE analysis of steps of additional purification of BBIonly. ST=standard (see FIG. 5(A-C) legend), 2 mg/ml. Lanes: 1: BBI poolfrom column. 2: Permeate of 30 KDa filtration. 3: Retentale of 30 KDafiltration diluted 1:40. 4: Retentate of 5 KDa concentration diluted1:4.

FIG. 7. SDS-PAGE analysis of 1 mg/ml. of the final product ofpurification of BBI only. ST=standard (see FIG. 5 legend), 1 mg/ml.

FIG. 8. Flowchart of revised protocol for downstream purification of BBIand KTI.

FIG. 9. Column chromatography run report using alternative procedure.

FIG. 10. SDS-PAGE analysis of duplicate samples of BBI purified usingalternative procedure (lanes 1-2) vs. standards (lane 3).

FIG. 11. Testing results of various emulsifier formulations. Foambuildup score was from 1-5, where 1 indicates no foam, and 5 indicatesno liquid visible because of the foam. For the suspension test, numbers1-5 indicate full phase separation; partial phase separation with somelarger oil bubbles; small oil bubble, milky consistency; no bubblesinitially, with later phase separation; and stable emulsion,respectively.

FIG. 12(A)-1 to FIG. 12(A)-4; FIG. 12(B)-1; and FIG. 12(B)-2. Bloodglucose profiles following administration of oral insulin formulationscontaining various emulsifiers. FIG. 12(A)-1. Formulation A (upperleft), FIG. 12(A)-2. Formulation B (lower left), FIG. 12(A)-3.Formulation C (upper right), and FIG. 12(A)-4. Formulation D (lowerright). FIG. 12(B)-1. Formulation E (left) and FIG. 12(B)-2. FormulationF (right).

FIG. 13(A) and FIG. 13(B): Patient record sheets. FIG. 13(A). BloodSugar Record. FIG. 13(B). Questionnaire.

FIG. 14. Hypoglycemia Review Sheet Points are awarded for eachoccurrence of documented hypoglycemia, with extra points given dependingon the neuroglycopenic symptoms experienced or lack thereof. Examplesfor the definition of symptoms were as follows: visual, eyes won'tfocus, impaired vision, double vision; behavioral, unable to sleep,irritable, stressed out nervous, “want to sit down and do nothing”,other neurological, light-headed, dizzy, weakness, tiredness, sleepy,difficulty walking or speaking, slow responses, delayed motor skills,loss of balance; confusion, inability to perform simple math, feeling“out of it”. No points are awarded if there were autonomic symptoms thatgave adequate warning of impending hypoglycemia, even if someneuroglycopenic symptoms were also present. Additional points are givenfor the need for outside help to either recognize or treat the event.

FIG. 15. Glucose responses in subjects treated with various doses oforal insulin.

DETAILED DESCRIPTION OF THE EMBODIMENTS

In one aspect, a BBI isolated from a soybean product is provided,wherein the BBI is at least 85% pure as measured, in variousembodiments, by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), Brilliant Blue staining, or imagerquantitation. In certain embodiments, the soybean product is soy flour.

In yet another aspect is provided a BBI isolated from soy flour, whereinthe protein content of the BBI is greater than 95% by BCA (bicinchoninicacid) assay.

In yet another aspect is provided a BBI isolated from soy flour, whereinthe BBI contains less than 0.1% high-MW contaminants, for example asassessed by SDS-PAGE and imager quantitation.

In other embodiments, the ratio of the anti-trypsin activity to theanti-chymotrypsin activity present in the isolated BBI is between 1.5:1and 1:1 inclusive. In more specific embodiments, the ratio may bebetween 1.4:1-1.1:1, inclusive. In more specific embodiments, the ratiomay be between 1.35:1-1.2:1, inclusive. In more specific embodiments,the ratio may be 1.28:1.

Unless indicated otherwise, anti-chymotrypsin activity referred toherein is measured using chymotrypsin having an activity of 40 BTEEunits per mg. of chymotrypsin, and is expressed in mg. of chymotrypsininhibited per mg. of protein being tested. BTEE refers toN-Benzoyl-L-Tyrosine Ethyl Ester (see the directions for Sigma-AldrichProduct No. B6125).

Unless indicated otherwise, anti-trypsin activity referred to herein ismeasured using trypsin having an activity of 10,000 BAEE units per mg.of trypsin, and is expressed in mg. of trypsin inhibited per mg. ofprotein being tested. BAEE refers to Na-Benzoyl-L-Arginine Ethyl EsterSolution (see the directions for Sigma-Aldrich Product No. B4500). Forexample, in a typical assay, one unit corresponds to the amount ofinhibitor that reduces the trypsin activity by one benzoyl-L-arginineethyl ester unit (BAEE-U). One BAEE-U is the amount of enzyme thatincreases the absorbance at 253 nm by 0.001 per minute at pH 7.6 and 25°C. See, for example, K. Ozawa, M. Laskowski, 1966, J. Biol. Chem.241:3955; and Y. Birk, 1976, Meth. Enzymol. 45:700.

Those skilled in the art will appreciate that each of the above purityrequirements, regarding its protein content, level of contaminants, orpotency, is typically assessed prior to the BBI being mixed with one ormore other components of the pharmaceutical composition.

In an additional aspect, a KTI3 isolated from soy flour is provided,wherein the KTI3 is at least 85% pure as measured, in variousembodiments, by SDS-PAGE, Brilliant Blue staining, or imagerquantitation.

In yet another aspect is provided a KTI3 isolated from soy flour,wherein the protein content of the KTI3 is greater than 95% as measuredby BCA assay.

In yet another aspect is provided a KTI3 isolated from soy flour,wherein the KTI3 contains less than 0.1% high-MW contaminants, forexample as assessed by SDS-PAGE and imager quantitation.

Those skilled in the art will appreciate that each of the above purityrequirements, regarding its protein content, level of contaminants, orpotency, is typically assessed prior to the KTI3 being mixed with one ormore other components of the pharmaceutical composition.

In certain embodiments, the KTI3-containing pharmaceutical compositionfurther comprises a BBI that meets at least one of the above purityrequirements prior to its being mixed with one or more other componentsof the pharmaceutical composition.

In certain embodiments, the above-described pharmaceutical compositionsare formulated for oral administration. In more specific embodiments,the pharmaceutical composition further comprises a coating that resistsdegradation in the stomach. In even more specific embodiments, thecoating is a pH-sensitive capsule, or alternatively, is a soft gelatincapsule.

In other embodiments, the above-described pharmaceutical compositionsfurther comprise a therapeutic protein of up to 100 kilodaltons as anactive ingredient. In other embodiments, the active ingredient is anon-protein molecule that is sensitive to degradation or inactivation inthe human digestive tract. In another embodiment, an oral pharmaceuticalcomposition is provided, comprising an oil-based liquid formulation,wherein the oil-based liquid formulation comprises a therapeutic proteinof up to 100 kilodaltons (kDa), a chelator of divalent cations, and anisolated BBI. In other embodiments, the liquid formulation consistsessentially of a therapeutic protein of up to 100 kDa, a chelator ofdivalent cations, an isolated BBI, and an oil. In other embodiments, theliquid formulation consists essentially of a therapeutic protein of upto 100 kDa, a chelator of divalent cations, an isolated BBI, an oil, andan emulsifier. In other embodiments, the liquid formulation consistsessentially of a therapeutic protein of up to 100 kDa, a chelator ofdivalent cations, an isolated BBI, an oil, and two emulsifiers.

In another aspect is provided an oral pharmaceutical compositioncomprising an oil-based liquid formulation, wherein the oil-based liquidformulation comprises a therapeutic protein of up to 100 kDa, a chelatorof divalent cations, and a soybean-derived BBI, wherein said liquidformulation contains less than 0.05% of soybean-derived substanceshaving a MW of greater than 30,000.

In another aspect is provided an oral pharmaceutical compositioncomprising an oil-based liquid formulation, wherein the oil-based liquidformulation comprises a therapeutic protein of up to 100 kDa and achelator of divalent cations, and said liquid formulation has ananti-chymotrypsin activity of at least 50 mg chymotrypsin inhibited perml. of the liquid formulation. In other embodiments, the liquidformulation has an anti-chymotrypsin activity of at least 35, 40, 45, 55or 60 mg. chymotrypsin inhibited per ml. of the liquid formulation. Instill other embodiments, the liquid formulation has an anti-chymotrypsinactivity in the range of 35-70, 40-70, 45-70, 50-70, or 40-60 mg. ofchymotrypsin inhibited per ml. of the liquid formulation. In otherembodiments, the liquid formulation further comprises an anti-trypsinactivity of at least 25 mg. of trypsin inhibited per ml. of the liquidformulation. In other embodiments, the liquid formulation furthercomprises an anti-trypsin activity of at least 30, 35, 40, 45, or 50 mg.trypsin inhibited per ml. of the liquid formulation. Alternatively, theliquid formulation further comprises an anti-trypsin activity in therange of 25-50, 30-50, 35-50, 25-40, or 25-45 mg. trypsin inhibited perml. of the liquid formulation.

In another aspect is provided a method for making a pharmaceuticalcomposition, comprising the steps of (a) providing a preparation ofisolated BBI, a therapeutic protein of up to 100 kilodaltons, and achelator of divalent cations; and (b) mixing said isolated BBI,therapeutic protein, and chelator into an oil-based liquid formulation.Each of the embodiments described herein of the identity, purity, andpotency of isolated BBI may be incorporated into this method. Inaddition, each of the embodiments described herein of the otheringredients, and of additional ingredients that may be present, may beincorporated into this method. In other embodiments, a pharmaceuticalcomposition made by this method is provided.

In another aspect is provided a method for making a pharmaceuticalcomposition, comprising the step of mixing an isolated BBI, atherapeutic protein of up to 100 kilodaltons, and a chelator of divalentcations into an oil-based liquid formulation. Each of the embodimentsdescribed herein of the identity, purity, and potency of isolated BBImay be incorporated into this method. In addition, each of theembodiments described herein of the other ingredients, and of additionalingredients that may be present, may be incorporated into this method.In other embodiments, a pharmaceutical composition made by this methodis provided.

“Liquid” as used herein refers to a composition that has a viscositywithin the range of 1-1000 millipascal seconds, inclusive, at 20° C.Fish oil, for instance, is a liquid under ambient conditions. The terraincludes oil-based solutions, suspensions, and combinations thereof.

“Isolated” BBI as used herein refers to a preparation enriched in BBIrelative to other components. In more specific embodiments, BBI refersto Bowman-Birk inhibitor; Uniprot number P01055 [database accessed onJan. 28, 2013]).

A representative precursor sequence of BBI is:

(SEQ ID NO: 1) MVVLKVCLVL LFLVGGTTSA NLRLSKLGLL MKSDHQHSNDDESSKPCCDQ CACTKSNPPQ CRCSDMRLNS CHSACKSCICALSYPAQCFC VDITDFCYEP CKPSEDDKEN.

Of these 110 residues, residues 1-19 are the signal peptide, 20-39 are apropeptide, and the mature chain BBI chain is composed of residues40-110 (71 AA).

In various embodiments, the preparation of BBI utilized in the describedmethods and compositions is at least 85%, 90%, 92%, 94%, or 95% pure asassessed by SDS-PAGE, Brilliant Blue staining, or imager quantitation(e.g. according to the protocol described herein). In the context of apharmaceutical composition, this value refers to characteristics of theBBI prior to its being mixed with one or more other components of thepharmaceutical composition. In alternative embodiments, SDS-PAGE andsilver staining, optionally followed by imager quantitation, may beutilized.

In other embodiments, the isolated BBI has an anti-chymotrypsin activityof at least 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, or 1.4 mg. chymotrypsininhibited per mg. inhibitor. In other embodiments, the anti-chymotrypsinactivity is in the range of 0.8-1.8, 0.9-1.8, 1.0-1.8, 1.1-1.8, 1.2-1.8,1.3-1.8, or 1.4-1.8 mg. chymotrypsin inhibited per mg. inhibitor. Inmore specific embodiments, the activity is 0.8-1.8 mg. chymotrypsininhibited per mg. inhibitor, in other embodiments, the activity is inthe range of 0.8-1.5 mg. chymotrypsin inhibited per mg. inhibitor. Inthe context of a pharmaceutical composition, this value refers tocharacteristics of the BBI prior to its being mixed with one or moreother components of the pharmaceutical composition.

In various embodiments, the BBI preparation contains 5% or less KTI asassessed by SDS-PAGE, Brilliant Blue staining, or imager quantitation(according to the protocol described herein). In alternativeembodiments, SDS-PAGE and silver staining may be utilized.

In more specific embodiments, KTI as used herein refers to KTI3 (Uniprotnumber P01070; database accessed on Jan. 3, 2013). A representativeprecursor sequence of KTI3 is:

SEQ ID NO: 2) MKSTIFFLFL FCAFTTSYLP SAIADFVLDN EGNPLENGGTYYILSDITAF GGIRAAPTGN ERCPLTVVQS RNELDKGIGTIISSPYRIRF IAEGHPLSLK FDSFAVIMLC VGIPTEWSVVEDLPEGPAVK IGENKDAMGD WFRLERVSDD EFNNYKLVFCPQQAEDDKCG DIGISIDHDD GTRRLVVSKN KPLVVQFQKL DKESLAKKNH GLSRSE

Of the above sequence, residues 1-24 are the signal peptide, 206-216 arethe propeptide, and the mature KTI chain is composed of residues 25-205(181 AA).

In other embodiments, the protein content of the BBI preparation isgreater than 95% by BCA assay (for example using the Micro BCA ProteinAssay Kit [cat #23225, Thermo Scientific Rockford, Ill.]). In thecontext of a pharmaceutical composition, this value refers to itscharacteristics prior to its being mixed with one or more othercomponents of the pharmaceutical composition. In other embodiments, theBBI preparation contains less than 0.1% high-MW contaminants (in otherwords, substances having a MW of greater than 30,000). In otherembodiments, the BBI has been isolated without the use ofpolyethyleneglycol (PEG).

In other embodiments, the isolated BBI in the described methods andcompositions is a recombinant BBI, for example BBI produced by amicroorganism such as a bacterium that has been engineered to express itand subsequently isolated. In still other embodiments, the BBI is asynthetic BBI. An example of a synthetic BBI is BBI that has beenproduced in a cell-free apparatus such as a peptide synthesizer. Peptidesynthesizers, for example automated peptide synthesizers, are well knownin the art and are available commercially. Pharmaceutical compositionscomprising recombinant BBI are also provided herein. Pharmaceuticalcompositions comprising synthetic BBI are also provided herein.

In certain embodiments, the described BBI is the only protease inhibitorin the described methods and compositions. While lower-potency SBTIrequires an additional protease inhibitor, e.g. aprotinin, toefficiently protect certain therapeutic proteins in the human digestivetract, the described isolated BBI is believed to be capable of reducingthe need for additional protease inhibitors in this regard.

Additional Protease Inhibitors

In certain embodiments, the oil-based liquid formulation utilized in thedescribed methods and compositions further comprises a trypsin inhibitorother than the isolated BBI. In other embodiments, the oil-based liquidformulation utilized in the described methods and compositions furthercomprises a trypsin inhibitor other than the isolated KTI3. Thoseskilled in the art will appreciate in light of the present disclosurethat a variety of trypsin inhibitors may be utilized. In the case oftrypsin inhibitors that are proteins, the size will typically be up to100 kDa.

As used herein, the term “trypsin inhibitor” refers to any agent capableof inhibiting the action of trypsin on a substrate. The ability of anagent to inhibit trypsin can be measured using assays well known in theart.

Some trypsin inhibitors known in the art are specific to trypsin, whileothers inhibit trypsin and other proteases such as chymotrypsin. Trypsininhibitors can be derived from animal or vegetable sources: for example,soybean, corn, lima and other beans, squash, sunflower, bovine and otheranimal pancreas and lung, chicken and turkey egg white, soy-based infantformula, and mammalian blood. Trypsin inhibitors can also be ofmicrobial origin: for example, antipain; see, for example, H. Umezawa,1976, Meth. Enzymol. 45, 678. A trypsin inhibitor can also be anarginine or lysine mimic or other synthetic compound: for examplearylguanidine, benzamidine, 3,4-dichloroisocoumarin,diisopropylfluorophosphate, gabexate mesylate, or phenylmethanesulfonylfluoride. As used herein, an arginine or lysine mimic is a compound thatis capable of binding to the P¹ pocket of trypsin and/or interferingwith trypsin active site function.

In certain embodiments, the additional trypsin inhibitor utilized in thedescribed methods and compositions is selected from the group consistingof lima bean trypsin inhibitor, aprotinin, (a.k.a. pancreatic trypsininhibitor or basic pancreatic trypsin inhibitor [BPTI]; Uniprot No.P00974 [database accessed on Jan. 2, 2013]), Kazal inhibitor (pancreaticsecretory trypsin inhibitor), Kazal inhibitor (pancreatic secretorytrypsin inhibitor), ovomucoid. Alpha 1-antitrypsin, Cortisol bindingglobulin, Centerin ([SERPINA9/GCET1 (germinal centre B-cell-expressedtranscript 1]), PI-6 (Sun et al 1995), PI-8 (Sprecher et al 1995),Bomapin, a clade A serpin [for example Serpina3 (NCBI Gene ID: 12),Serpina6 (NCBI Gene ID: 866), Serpina12 (NCBI Gene ID: 145264);Serpina10 (NCBI Gene ID: 51156); Serpina7 (NCBI Gene ID: 6906); Serpina9(NCBI Gene ID: 327657); Serpina11 (NCBI Gene ID: 256394); Serpina13(NCBI Gene ID: 388007); Serpina2 (NCBI Gene ID: 390502); and Serpina4(NCBI Gene ID: 5104)] Yukopin (Serpinb12; Gene ID: 89777), antipain,benzamidine, 3,4-dichloroisocoumarin, diisopropylfluorophosphate, andgabexate mesylate. In other embodiments, one of the above inhibitors isselected.

A representative precursor sequence of aprotinin is:

(SEQ ID NO: 3) MKMSRLCLSV ALLVLLGTLA ASTPGCDTSN QAKAQRPDFCLEPPYTGPCK ARIIRYFYNA KAGLCQTFVY GGCRAKRNNF KSAEDCMRTC GGAIGPWENL.

Of these 100 residues, residues 1-21 are the signal peptide, 22-35 and94-100 are propeptides, and the mature chain BBI chain is composed ofresidues 36-93 (58 AA).

In other embodiments, an oil-based liquid formulation utilized in thedescribed methods and compositions comprises both isolated BBI andisolated KTI, in more specific embodiments both isolated BBI andisolated KTI3. “Isolated” KTI as used herein refers to a preparationenriched in KTI relative to other components. In various embodiments,the preparation of KTI utilized in the described methods andcompositions is at least 85% pure as assessed by SDS-PAGE, BrilliantBlue staining, or imager quantitation (e.g. according to the protocoldescribed herein), in other embodiments, the protein content of the KTIpreparation is greater than 95% by BCA assay. In the context of apharmaceutical composition, these values refer to characteristics of theKTI prior to its being mixed with one or more other components of thepharmaceutical composition. In other embodiments, the KTI preparationcontains 5% or less BBI as assessed by SDS-PAGE. In other embodiments,the KTI preparation contains less than 0.1% high-MW contaminants (inother words, substances having a MW of greater than 30,000). In otherembodiments, the KTI has been isolated without the use of PEG.

In still more specific embodiments, the described methods andcompositions comprise the described BBI and KTI, in more specificembodiments BBI and KTI3, as the only protease inhibitors. In otherembodiments, the described methods and compositions comprise KTI andaprotinin, in more specific embodiments KTI3 and aprotinin, as the onlyprotease inhibitors. In other embodiments, isolated BBI, isolated KTI,and aprotinin are all present in the oil-based liquid formulation.

In other embodiments, the isolated KTI3 has an activity of at least 0.8,0.9, 1.0, 1.1, 1.2, or 1.3 mg. trypsin inhibited per mg. inhibitor. Inother embodiments, the activity of the KTI3 is in the range of 0.8-1.8,0.9-1.8, 1.0-1.8, 1.1-1.8, 1.2-1.8, or 1.3-1.8 mg. trypsin inhibited permg. inhibitor. In more particular embodiments, the activity of the KTI3is 0.8-1.7 mg. trypsin inhibited per mg. inhibitor. In otherembodiments, the activity is range of 0.8-1.4 mg. trypsin inhibited permg. inhibitor.

In other preferred embodiments, the isolated KTI is a recombinant KTI,for example KTI produced by a microorganism such as a bacterium that hasbeen engineered to express it. In still other preferred embodiments, theKTI is a synthetic KTI. An example of a synthetic KTI is KTI that hasbeen produced in a cell-free apparatus such as a peptide synthesizer.

Other embodiments concern the ratio of the anti-chymotrypsin activitypresent in the described pharmaceutical composition to the anti-trypsinactivity of the composition. In some embodiments, this parameter isbetween 1.5:1 and 1:1 inclusive. In more specific embodiments, the ratiomay be between 1.4:1-1.1:1, inclusive. In more specific embodiments, theratio may be between 1.35:1-1.2:1, inclusive.

In certain embodiments, the BBI utilized in the described methods andcompositions, and/or KTI, if present, has been stored with apreservative. In other embodiments, the BBI and/or KTI has been preparedand stored without use of a preservative.

In certain embodiments, the BBI utilized in the described methods andcompositions, and/or the KTI, if present, is obtained from soy flour.The term “soy flour” refers to flour obtained from the species Glycinemax. In other embodiments, any species from the genus Glycine may beutilized. Methods for obtaining soy flour ere well known in the art. Thedescribed methods are believed be applicable to any type of soy flour,no matter how it was produced.

Therapeutic Proteins

Therapeutic proteins for compositions and methods described herein arein some embodiments isolated prior to inclusion in the describedpharmaceutical compositions. “Isolated” in this regard excludesprovision of the therapeutic protein as a homogenized tissue preparationor other form containing substantial amounts of contaminating proteins.A preferred example of an isolated protein or peptide is a recombinantprotein or peptide. An even more preferred embodiment is a syntheticprotein, in other words a protein produced in a cell-free apparatus.Those skilled in the art will appreciate in light of the presentdisclosure that both wild-type and mutated therapeutic proteins may beutilized.

Certain proteins and peptides are known to be specific inhibitors oftrypsin and/or chymotrypsin, including but not limited to thosedescribed herein as being trypsin and/or chymotrypsin inhibitors. Suchproteins are not intended for use as the therapeutic component in thedescribed compositions, and are excluded from the definition of“therapeutic proteins” as used herein.

Those of skill in the art will appreciate in light of the presentdisclosure that a variety of therapeutic proteins may be used in thedescribed methods and compositions. In certain embodiments, thetherapeutic protein is up to 100 kilodaltons (kDa) in size, typicallybetween 1-100 kDa, inclusive. In more specific embodiments, the size isup to 90 kDa. In other embodiments, the size is up to 80 kDa. In otherembodiments, the size is up to 70 kDa. In other embodiments, the size isup to 60 kDa. In other embodiments, the size is up to 50 kDa.Preferably, the size is between 1-90 kDa, inclusive. In otherembodiments, the size is between 1-80 kDa, inclusive. In otherembodiments, the size is between 1-70 kDa, inclusive. In otherembodiments, the size is between 1-60 kDa, inclusive. In otherembodiments, the size is between 1-50 kDa, inclusive.

Therapeutic proteins suitable for use herein include derivatives thatare modified (i.e., by the covalent attachment of a non-amino acidmoiety to the protein). For example, but not by way of limitation, theprotein includes proteins that have been modified, e.g., byglycosylation, acetylation, PEGylation, phosphorylation, amidation, orderivatization by known protecting/blocking groups. High-MW PEG can beattached to therapeutic proteins with or without a multifunctionallinker either through site-specific conjugation of the PEG to the N- orC-terminus thereof or via epsilon-amino groups present on lysineresidues. Additionally, the derivative may contain one or morenon-classical amino acids.

In certain, more specific, embodiments, the therapeutic protein utilizedin the described methods and compositions is selected from the groupconsisting of insulin, influenza hemagglutinin, influenza neuraminidase,glucagon, interferon gamma, interferon beta, interferon alpha, growthhormone, erythropoietin, GLP-1, a GLP-1 analogue, granulocyte colonystimulating factor (G-CSF), renin, growth hormone releasing factor,parathyroid hormone, thyroid stimulating hormone, follicle stimulatinghormone, calcitonin, luteinizing hormone, glucagon, a clotting factor(for example factor VII, factor VIIIC, factor DC, tissue factor (TF),and thrombin), an anti-clotting factor (for example Protein C), atrialnatriuretic factor, surfactant protein A (SP-A), surfactant protein B(SP-B), surfactant protein C (SP-C), surfactant protein D (SP-D), aplasminogen activator (for example urokinase or human urine ortissue-type plasminogen activator (t-PA)), bombesin, hemopoietic growthfactor (a.k.a. colony-stimulating factor, multiple), a tumor necrosisfactor (TNF) protein (for example TNF-alpha, TNF-beta, TNF beta-2,4-1BBL), enkephalinase, RANTES (regulated on activation normally T-cellexpressed and secreted), human macrophage inflammatory protein(MIP-1-alpha), serum albumin, Mullerian-inhibiting substance, relaxin,mouse gonadotropin-releasing hormone, DNase, inhibin, activin, vascularendothelial growth factor (VEGF), a neurotrophic factor (for examplebrain-derived neurotrophic factor [BDNF]), neurotrophin-3, -4, -5, or -6(NT-3, NT-4, NT-5, or NT-6), nerve growth factor, platelet-derivedgrowth factor (PDGF), a fibroblast growth factor (for example alpha-FGFand beta-FGF), a transforming growth factor (TGF) (for example TGF-alphaand TGF-beta, including TGF-1, TGF-2, TGF-3, TGF-4, and TGF-5),insulin-like growth factor-I and -II (IGF-I and IGF-II), des (1-3)-IGF-I(brain IGF−1), insulin-like growth factor binding proteins (includingIGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6), akeratinocyte growth factor, an osteoinductive factor, bone morphogeneticprotein (BMP)-2, BMP-7, a colony stimulating factor (CSF) (for exampleM-CSF and GM-CSF), an interleukin (IL), (for example IL-1 to IL-13 andIL-15, IL-18, and IL-23), superoxide dismutase, decay acceleratingfactor, a chemokine family member (for example the eotaxins and MCP-1),and a complement factor (for example C3 and C5).

In still more specific embodiments, the therapeutic protein is insulin.Alternatively, the therapeutic protein may be a GLP-1 inhibitor. In amore specific embodiment, the therapeutic protein is exenatide. In otherembodiments, both insulin and exenatide are present in the describedcomposition. In other embodiments, the liquid formulation consistsessentially of insulin, exenatide, a chelator of divalent cations, anisolated BBI, and an oil. In other embodiments, the liquid formulationconsists essentially of insulin, exenatide, a chelator of divalentcations, an isolated BBI, at least one emulsifier, and an oil. In stillother embodiments, the liquid formulation consists essentially ofinsulin, exenatide, a chelator of divalent cations, an isolated KTI3,aprotinin, and an oil. In yet embodiments, the liquid formulationconsists essentially of insulin, exenatide, a chelator of divalentcations, an isolated KTI3, aprotinin, at least one emulsifier, and anoil.

A person skilled in the art will appreciate in light of the presentdisclosure that various types of insulin are suitable for the describedmethods and compositions. Exemplary insulin proteins include but are notlimited to both wild-type and mutated insulin proteins, includingsynthetic human insulin, synthetic bovine insulin, synthetic porcineinsulin, synthetic whale insulin, and metal complexes of insulin, suchas zinc complexes of insulin, protamine zinc insulin, and globin zinc.

Various classes of insulin may also be utilized, for example fast-actinginsulin, lente insulin, semilente insulin, ultralente insulin, NPHinsulin, glargine insulin, lispro insulin, aspart insulin, orcombinations of two or more of the above types of insulin.

In a particularly preferred embodiment, the insulin of the describedmethods and compositions is wild-type human insulin (Uniprot ID P01308;SEQ ID NO: 4). Of the 110 amino acids, 1-24 is the signal peptide, 25-54forms the insulin B chain, 57-87 forms C peptide, and 90-110 forms theinsulin A chain. In one preferred embodiment, human insulin is producedas a recombinant protein in bacterial cells. In another preferredembodiment, human insulin is produced synthetically.

GLP-1 analogues are also referred to in the art as GLP-1 mimetics. Aperson of skill in the art will appreciate in light of the presentdisclosure that the described compositions may include at least one ofthe following GLP-1 analogues: exenatide (Byetta™; CAS no. 141732-76-5;SEQ ID NO: 5), lixisenatide (CAS no. 320367-13-3), liraglutide (CAS no.204656-20-2), exendin-9 (CAS no. 133514-43-9), AC3174([Leu(14)]exendin-4, Amylin Pharmaceuticals, Inc.), taspoglutide (CASno. 275371-94-3), albiglutide (CAS no. 782500-75-8), semaglutide (CASno. 910463-68-2), LY2189265 (Dulaglutide™; CAS no. 923950-08-7), andCJC-1134-PC (a modified Exendin-4 analogue conjugated to recombinanthuman albumin manufactured by ConjuChem™). All CAS records were accessedon Dec. 19, 2011. Thus, in certain embodiments, the described method orcomposition utilizes any of the above-listed GLP-1 analogues. In otherembodiments, one of the above-listed GLP-1 analogues is selected. Thoseof skill in the art will appreciate in light of the findings presentedherein that other GLP-1 analogues can also be utilized in the describedmethods and compositions.

Emulsifiers

“Weight/weight” percentages of emulsifiers and detergents referred toherein utilize the amount of oil base in the formulation, for examplefish oil, as the denominator; thus, 60 mg of Gelucire in 500 mg fish oilis considered as 12% w/w, regardless of the weight of the othercomponents. Similarly, 50 mg. Tween-80 mixed with 500 mg fish oil isconsidered as 10% Tween-80.

In certain embodiments, the oil-based liquid formulation utilized in thedescribed methods and pharmaceutical compositions, or in otherembodiments, each of the oil-based liquid formulation that is present,comprises, in addition to the therapeutic protein, chelator, and BBI, apolyethylene glycol (PEG) ester of a fatty acid, for example a PEG esterof a monoglyceride, a diglyceride, a triglyceride, or a mixture thereof.In more specific embodiments, the PEG ester may be provided as a mixtureof (a) a free monoacylglycerol, a free diacylglycerol, a treetriacylglycerol, or a mixture thereof; and (b) a PEG ester of a fattyacid, for example a PEG ester of a monoglyceride, a diglyceride, atriglyceride, or a mixture thereof. In this regard, each of the terms“monoacylglycerol”, “diacylglycerol”, and “triacylglycerol” need notrefer to a single compound, but rather can include mixtures ofcompounds, for example mixtures of monoacylglycerols, diacylglycerols,or triacylglycerols having fatty acids of varying lengths.

In certain preferred embodiments, monoacylglycerols, diacylglycerols, ortriacylglycerols utilized in the described methods and compositions, forexample those used to general PEG esters, are from an oil source that isGenerally Recognized As Safe (GRAS). Examples of GRAS oils are coconutoil, corn oil, peanut oil, soybean oil, Myvacet 9-45 (Diacetylatedmonoacylglycerols of C-18 forty acids). A more specific embodiment of(a) is a mixture of C₈-C₁₈ monoacylglycerols, diacylglycerols, andtriacylglycerols. A more specific embodiment of component (b) is amixture of PEG monoesters and diesters of one or more C₈-C₁₈ fattyacids.

In more specific embodiments, the liquid formulation further comprises,in addition to the PEG ester of a fatty acid, a free PEG. In still morespecific embodiments, an additional non-ionic detergent, for example apolysorbate-based detergent, is present in addition to the PEG ester andfree PEG.

In a still more specific embodiment of the described methods andcompositions, a liquid formulation comprises: (a) a mixture of C₈-C₁₈monoacylglycerols, diacylglycerols, and triacylglycerols; (b) PEG-32monoesters and diesters of a mixture of C₈-C₁₈ fatty acids; and (c) freePEG-32. In even more specific embodiments, the weight/weight ratio ofcomponent (a) to the sum of components (b)+(c) is between 10:90-30:70inclusive; more specifically between 15:85-25:75 inclusive; morespecifically 20:80. In certain embodiments, components (a)-(c) togetherconstitute 8-16% weight/weight inclusive of the oil-based liquidformulation. In more specific embodiments, the amount is 9-15%inclusive. In more specific embodiments, the amount is 10-14% inclusive.In more specific embodiments, the amount is 11-13% inclusive. In morespecific embodiments, the amount is 12%.

In other embodiments, an oil-based liquid formulation utilized in thedescribed methods and pharmaceutical compositions comprises, in additionto the therapeutic protein, chelator, and BBI, a self-emulsifyingcomponent. While some embodiments of self-emulsifying components are themixtures of components described in the preceding paragraphs, thesemixtures do not limit the definition of the term “self-emulsifyingcomponents” as used herein. “Self-emulsifying component” as used hereinrefers to a component that spontaneously forms an emulsion. Typically,such components will form an emulsion on contact with aqueous media,forming a fine dispersion i.e. a microemulsion (SMEDDS). Certainembodiments of such components comprise a triacylglycerol mixture oftriacylglycerols and a high hydrophile/lipophile balance (HLB; seeGriffin W C: “Calculation of HLB Values of Non-Ionic Surfactants,” J SocCosmetic Chemists 5:259 (1954)) surfactant. Other embodiments of theself-emulsifying component have a waxy, semi solid consistency.

Preferably, the HLB of a self-emulsifying component utilized in thedescribed methods and compositions is 10 or greater. In otherembodiments, it is between 11-19 inclusive. In other embodiments, it isbetween 12-18 inclusive. In other embodiments, it is between 12-17inclusive. In other embodiments, it is between 12-16 inclusive, which isindicative of an oil-in-water (O/W) emulsifier. In other embodiments, itis between 13-15 inclusive. In other embodiments, it is 14. Still morespecific embodiments of self-emulsifying components have an HLB of 12-16inclusive and comprise medium- and long-chain triacylglycerolsconjugated to PEG, free triacylglycerols, and free PEG. In otherembodiments, the self-emulsifying component has an HLB of 12-16inclusive and consists of a mixture of medium- and long-chaintriacylglycerols conjugated to PEG, free triacylglycerols, and free PEG.In other embodiments, the self-emulsifying component has an HLB of 14and comprises medium- and long-chain triacylglycerols conjugated to PEG,free triacylglycerols, and free PEG. In other embodiments, theself-emulsifying component has an HLB of 14 and consists of a mixture ofmedium- and long-chain triacylglycerols conjugated to PEG, freetriacylglycerols, and free PEG.

Certain, more specific embodiments utilize self-emulsifying componentsthat comprise (a) a monoacylglycerol, a diacylglycerol, atriacylglycerol, or a mixture thereof; and (b) a polyethylene glycol(PEG) ester of a fatty acid, in this regard, each of the terms“monoacylglycerol”, “diacylglycerol”, and “triacylglycerol” need notrefer to a single compound, but rather can include mixtures ofcompounds, for example mixtures of monoacylglycerols, diacylglycerols,or triacylglycerols having fatty acids of varying lengths. A morespecific embodiment is a mixture of C₈-C₁₈ monoacylglycerols,diacylglycerols, and triacylglycerols. A more specific embodiment ofcomponent (b) is a mixture of PEG monoesters and diesters of a mixtureof C₈-C₁₈ fatty acids.

In other, more specific embodiments, the self-emulsifying componentfurther comprises free PEG.

Certain PEG moieties for use in the described compositions and methodscontain between 5-100 monomers. In more specific embodiments, the PEGmay contain between 15-50 monomers. In still more specific embodiments,the PEG may contain between 25-40 monomers. In more specificembodiments, the PEG may contain 32 monomers.

In still more specific embodiments of the described methods andcompositions, a self-emulsifying component used therein comprises: (a) amixture of C₈-C₁₈ monoacylglycerols, diacylglycerols, andtriacylglycerols; (b) PEG-32 monoesters and diesters of a mixture ofC₈-C₁₈ fatty acids; and (c) free PEG-32; and the weight/weight ratio ofcomponent (a) to components (b)+(c) is 20:80. In certain embodiments,such a component constitutes 8-16% weight/weight inclusive of theoil-based liquid formulation. In more specific embodiments, the amountis 9-15% inclusive, in more specific embodiments, the amount is 10-14%inclusive. In more specific embodiments, the amount is 11-13% inclusive.In more specific embodiments, the amount is 12%.

Examples of self-emulsifying components meeting the above specificationsare Gelucire™ 44/14, Gelucire™ 53/10, and Gelucire™ 50/13. A morespecific embodiment is Gelucire™44/14. The suffixes 44 and 14 referrespectively to its melting point and its hydrophilic/lypophilic balance(HLB). Gelucire™ 44/14 (Gattefossé SAS, Saint-Priest, France) isobtained by polyglycolysis of hydrogenated coconut oil (medium- andlong-chain triacylglycerols with PEG-32. It has a hydrophile/lipophilebalance of 14. It is composed of a defined admixture of C₈-C₁₈ mono-,di- and triacylglycerols (20% w/w); PEG-32 mono- and diesters and freePEG-32 (80% w/w). The main fatty acid present is lauric acid, accountingfor 45% on average of the total fatty acid content. It is a soliddispersion composed of a PEG ester fraction under a lamellar phase of120 Å with a helical conformation and an acylglycerol fraction under ahexagonal packing. The main products of simulated gastrointestinallipolysis of Gelucire™ 44/14 are PEG-32 mono and diesters. In morespecific embodiments, the described compositions comprise about 12%Gelucire 44/14 as the only emulsifier, or, in other embodiments,together with another emulsifier. In other embodiments, the describedcompositions comprise about 12% Gelucire 44/14 and about 10% Tween-80.

Non-ionic Detergents

In certain embodiments, an oil-based liquid formulation utilized in thedescribed methods and pharmaceutical compositions further comprises anon-ionic detergent in addition to the self-emulsifying component. Incertain embodiments, the non-ionic detergent is selected from the groupconsisting of polysorbate-20, polysorbate-40, polysorbate-80,lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenatedcastor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65and 80, sucrose fatty acid ester, methyl cellulose, carboxymethylcellulose, n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton™-X-100,Triton™-X-114, Thesit™, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), and N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate. Inother embodiments, one of the above-listed non-ionic detergents isselected.

In certain, more specific embodiments, a non-ionic detergent used in thedescribed methods and compositions is a polysorbate-based detergent.Examples of polysorbate-based detergents are detergents derived bycovalently bonding polyethoxylated sorbitan to a fatty acid. Morespecific embodiments of polysorbate-based detergents are polysorbate-20,polysorbate-40, and polysorbate-80.

For example, polysorbate 80 (Tween-80) is a non-ionic detergent derivedfrom polyethoxylated sorbitan and oleic acid and having the followingstructure:

In the case of polysorbate 80, the moiety shown on the right side is amixture of fatty acids, containing 60-70% oleic acid (as depicted), withthe balance being primarily linoleic, palmitic, and stearic acids.

In a more specific embodiment, the polysorbate 80 constitutes 3-15%weight/weight inclusive of an oil-based liquid formulation used in thedescribed methods and compositions. In a more specific embodiment, thepercentage is 5-14% inclusive. In a more specific embodiment, thepercentage is 7-12% inclusive. In more specific embodiments, thepercentage is 10%, or alternatively 5%.

Chelators of Divalent Cations

The chelator of divalent cations utilized in the described methods andcompositions is, in one embodiment, any physiologically acceptablecompound having a high affinity for at least one of calcium, magnesium,and manganese ions. In another embodiment, the chelator is selected fromthe group consisting of citrate or a salt thereof, ethylenediaminetetracetic acid (EDTA) or a salt thereof (for example disodium EDTA andcalcium disodium EDTA); EGTA (ethylene glycol tetraacetic acid) or asalt thereof; diethylene triamine pentaacetic acid (DTPA) or a saltthereof; and BATA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraaceticacid) or a salt thereof. In other embodiments, one of the above-listedchelators is utilized. In more specific embodiments, the chelator isEDTA.

Oils

Those of skill in the art will appreciate, in light of the presentfindings, that various oils may be utilized as the basis of their liquidphase of the described compositions. In certain embodiments, the oil maybe any physiologically acceptable oil that is liquid at ambienttemperature.

In more specific embodiments, the oil comprises an omega-3 fatty acid.In other embodiments, the omega-3 fatty acid is an omega-3polyunsaturated fatty acid. In another embodiment, the omega-3 fattyacid is DHA, an omega-3, polyunsaturated, 22-carbon fatty acid alsoreferred to as 4, 7, 10, 13, 16, 19-docosahexaenoic acid. In anotherembodiment, the omega-3 fatty acid is -linolenic acid (9, 12,15-octadecatrienoic acid), in another embodiment, the omega-3 fatty acidis stearidonic acid (6, 9, 12, 15-octadecatetraenoic acid). In anotherembodiment, the omega-3 fatty acid is eicosatrienoic acid (ETA; 11, 14,17-eicosatrienoic acid). In another embodiment, the omega-3 fatty acidis eicosatetraenoic acid (8, 11, 14, 17-eicosatetraenoic acid). In oneembodiment, the omega-3 fatty acid is eicosapentaenoic acid (EPA; 5, 8,11, 14, 17-eicosapentaenoic acid). In another embodiment, the omega-3fatty acid is eicosahexaenoic acid (also referred to as 5, 7, 9, 11, 14,17-eicosahexaenoic acid). In another embodiment, the omega-3 fatty acidis docosapentaenoic acid (DPA; 7, 10, 13, 16, 19-docosapenatenoic acid).In another embodiment, the omega-3 fatty acid is tetracosahexaenoic acid(6, 9, 12, 15, 18, 21-tetracosahexaenoic acid).

In other embodiments, the oil is a naturally-occurring oil comprising anomega-3 fatty acid. In more specific embodiments, the oil is selectedfrom the group consisting of a fish oil, canola oil, flaxseed oil, algaloil and hemp seed oil. In more specific embodiments, the oil is a fishoil. Several types of fish oil have been tested in the describedcompositions and have all been found to work equally well.

In other embodiments, a liquid formulation utilized in the describedmethod or composition is water-free. “Water-free” refers, in certainembodiments, to a formulation into which no aqueous components have beenintentionally added. It does not preclude the presence of trace amountsof water that have been absorbed from the atmosphere into the componentsthereof. In another embodiment, the liquid formulation is free ofaqueous components. In yet other embodiments, one or more oils are theonly liquid components of the liquid formulation. In more specificembodiments, fish oil is the only liquid component of the liquidformulation.

Coatings

Those of skill in the art will appreciate, in light of the presentfindings, that various pH-sensitive coatings may be utilized in thedescribed methods and compositions. In certain embodiments, any coatingthat inhibits digestion of the composition in the stomach of a subjectmay be utilized.

In other embodiments, the coating comprises a biodegradablepolysaccharide. In other embodiments, a hydrogel is utilized. In otherembodiments, the coating comprises one of the following excipients:chitosan, an aquacoat ECD coating, an azo-crosslinked polymer, celluloseacetate phthalate, cellulose acetate trimellitate (CAT), celluloseacetate butyrate, hydroxypropylmethyl cellulose phthalate, or poly vinylacetate phthalate.

In other embodiments, a timed-release system such as Pulsincap™ isutilized.

In preferred embodiments, the described coated dosage forms release thecore (containing the oil-based formulation) when pH reaches the rangefound in the intestines, which is alkaline relative to that in thestomach. In more specific embodiments, the coating comprises apH-sensitive polymer. In various embodiments, either mono-layer ormulti-layer coatings may be utilized.

In one embodiment, the coating is an enteric coating. Methods forenteric coating are well known in the art (sec, for example, Siepmann Fet al 2005). In more specific embodiments, a Eudragit™ coating isutilized as the enteric coating. Eudragit™ coatings are acrylicpolymers, the use of which is well known in the art.

In another embodiment, microencapsulation is used as a stomach-resistantcoating in the described compositions. Methods for microencapsulationare well known in the art.

In other embodiments, the coating is a capsule, of which gelatincapsules are a more specific embodiment. Methods for inserting anoil-based formulation into a gelatin capsule are well known in the art.In still other embodiments, the coating is a soft-gel, enteric-coatedcapsule.

In another embodiment, an oral pharmaceutical composition is provided,comprising an oil-based liquid formulation, wherein the oil-based liquidformulation comprises a therapeutic protein of up to 100 kilodaltons, achelator of divalent cations, an isolated chymotrypsin/trypsininhibitor, an isolated trypsin inhibitor, and a PEG ester of a fattyacid. In other embodiments, the liquid formulation has ananti-chymotrypsin activity of at least 50 mg. chymotrypsin inhibited perml. of the liquid formulation, in other embodiments, the liquidformulation has both an anti-chymotrypsin activity of at least 50 mg.chymotrypsin inhibited per ml. of the liquid formulation and ananti-trypsin activity of at least 25 trypsin inhibited per ml. of theliquid formulation. In other embodiments, a free PEG is also present. Inother embodiments, a non-ionic detergent is also present. In otherembodiments, the liquid formulation consists essentially of atherapeutic protein of up to 100 kilodaltons, a chelator of divalentcations, a chymotrypsin/trypsin inhibitor, a trypsin inhibitor, and aPEG ester of a fatty-acid. In other embodiments, the liquid formulationconsists essentially of a therapeutic protein of up to 100 kilodaltons,a chelator of divalent cations, a chymotrypsin/trypsin inhibitor, atrypsin inhibitor, a PEG ester of a fatty acid, and a free PEG. In otherembodiments, the liquid formulation consists essentially of atherapeutic protein of up to 100 kilodaltons, a chelator of divalentcations, a chymotrypsin/trypsin inhibitor, a trypsin inhibitor, a PEGester of a fatty acid, a free PEG, and a non-ionic detergent

Representative Formulations

Certain representative insulin formulations comprise insulin, Gelucire44/14, EDTA, SBTI, and aprotinin in fish oil, coated in a capsule.Certain representative exenatide formulations contain exenatide,Gelucire 44/14, EDTA, SBTI, and aprotinin in fish oil, coated in acapsule. One more specific embodiment is a formulation having thefollowing components: 8-20% Gelucire 44/14; 50-100 mg. per capsuleisolated BBI, or isolated BBI/isolated KTI mixture; 20-30 mg. percapsule Aprotinin; and 100-200 mg EDTA; with a therapeutic protein,which may be 8-32 mg. per capsule insulin and/or 150-600 mcg. percapsule Exenatide, all combined into 0.5-0.7 ml. fish oil. Anotherrepresentative liquid insulin formulation contains insulin, Gelucire44/14, EDTA, BBI, KTI, and aprotinin in fish oil. In other embodiments,the liquid formulation consists essentially of insulin, Gelucire 44/14,EDTA, BBI, KTI, aprotinin, and fish oil. More specific formulationscontain 8 mg insulin, 12% Gelucire 44/14, 150 mg EDTA, 75 mg total ofBBI and KTI, and 24 mg aprotinin in 0.5-0.7 ml. fish oil; 16 mg insulin,12% Gelucire 44/14, 150 mg EDTA, 75 mg total of BBI and KTI, and 24 mgaprotinin in 0.5-0.7 ml. fish oil; and 16 mg insulin, 12% Gelucire44/14, 150 mg EDTA, 150 mg total of BBI and KTI, and 24 mg aprotinin in0.5-0.7 ml. fish oil. In a still more specific formulation, the ratio ofthe anti-trypsin activity to the anti-chymotrypsin activity of thecomposition is about 1.28:1. In other embodiments, the above compositionfurther comprises a non-ionic detergent. In more specific embodiments,the non-ionic detergent is a polysorbate-based detergent, in even morespecific embodiments, the polysorbate-based detergent is polysorbate 80.Preferably, the polysorbate 80 constitutes 3-10% weight/weight inclusiveof the oil-based liquid formulation. The liquid formulation may becoated by a soft-gel, enteric-coated capsule. Specific formulationsdescribed in this paragraph also encompass, in certain embodiments,scaled-up and scaled-down formulation containing the same components inthe same ratios.

Some representative oral exenatide formulations comprise exenatide,EDTA, BBI, KTI, and aprotinin in fish oil. In other embodiments, theliquid formulation consists essentially of exenatide, Gelucire 44/14,EDTA, BBI, KTI, aprotinin, and fish oil. More specific formulationscontain 150 microgram (meg) exenatide, 150 mg EDTA, 75 mg total of BBIand KTI, and 24 mg aprotinin in 0.5-0.7 ml. fish oil; 300 meg exenatide,150 mg EDTA, 75 mg total of BBI and KTI, and 24 mg aprotinin in 0.5-0.7ml. fish oil; and 300 meg exenatide, 150 mg EDTA, 150 mg total of BBIand KTI, and 24 mg aprotinin in 0.5-0.7 ml. fish oil. In a still morespecific formulation, the ratio of the anti-trypsin activity to theanti-chymotrypsin activity of the composition is between 1.5:1 and 1:1,inclusive. In even more specific embodiments, the ratio is about 1.28:1.The liquid formulation may be coated by a soft-gel, enteric-coatedcapsule. In other embodiments, the above composition further comprises anon-ionic detergent. In more specific embodiments, the non-ionicdetergent is a polysorbate-based detergent. In even more specificembodiments, the polysorbate-based detergent is polysorbate 80.Preferably, the polysorbate 80 constitutes 3-10% weight/weight inclusiveof the oil-based liquid formulation. The liquid formulation may becoated by a soft-gel, enteric-coated capsule.

Therapeutic Indications

In another aspect is provided use of a BBI described herein in thepreparation of a medicament for orally administering an activeingredient to a subject. In another aspect is provided use of a KTI3described herein in the preparation of a medicament for orallyadministering an active ingredient.

In still another aspect is provided a method for making a pharmaceuticalcomposition, comprising the steps of: (a) producing a mixture comprisinga BBI described herein and an active ingredient; and (b) formulating themixture into a pharmaceutically acceptable formulation.

In still another aspect is provided a method for making a pharmaceuticalcomposition, comprising the steps of: (a) producing a mixture comprisinga KTI3 described herein and an active ingredient; and (b) formulatingthe mixture into a pharmaceutically acceptable formulation.

As referred to herein, the step of formulating comprises the steps ofoptionally adding excipients, milling, coating, and the like, asappropriate for the desired pharmaceutical composition. These steps arewell within the ability of those skilled in the art.

In certain embodiments, an active ingredient as referred to herein issensitive to degradation or inactivation in the human digestive tract.

In another aspect is provided an oral pharmaceutical compositiondescribed herein for orally administering an active ingredient to asubject. In certain preferred embodiments, the active ingredient issensitive to degradation or inactivation in the human digestive tract.In more specific embodiments, the active ingredient may be a therapeuticprotein. In other embodiments, the active ingredient is a non-proteinmolecule that is sensitive to degradation or inactivation in the humandigestive tract.

In another aspect is provided use of an oral pharmaceutical compositiondescribed herein in the preparation of a medicament for orallyadministering a therapeutic protein to a subject.

In another aspect is provided a method for orally administering atherapeutic protein to a subject, the method comprising the step ofadministering to a subject an oral pharmaceutical composition describedherein, thereby orally administering a therapeutic protein to a subject.

In another aspect is provided a pharmaceutical composition describedherein for treating diabetes in a human, where, in some embodiments, thetherapeutic protein is in various embodiments insulin, exenatide, or acombination thereof.

In yet another aspect is provided a use of a combination of ingredientsdescribed herein in the preparation of a medicament for treatingdiabetes in a human, where, in some embodiments, the therapeutic proteinis in various embodiments insulin, exenatide, or a combination thereof.

In still another aspect is provided a method for treating diabetes, themethod comprising the step of administering to a subject in need of suchtreatment a pharmaceutical composition described herein, where, in someembodiments, the therapeutic protein is in various embodiments insulin,exenatide, or a combination thereof, thereby treating diabetes. Incertain embodiments, the subject is a human subject, while in otherembodiments, the subject is a non-human mammal.

In an additional aspect is provided a pharmaceutical compositiondescribed herein for treating unstable diabetes in a human. In anotheraspect, a pharmaceutical composition described herein is provided fortreating an elevated fasting blood glucose level in a human.

In yet another aspect is provided a use of a combination of ingredientsdescribed herein in the preparation of a medicament for treatingunstable diabetes in a human. Additionally, a use is provided of acombination of ingredients described herein in the preparation of amedicament for treating an elevated fasting blood glucose level in ahuman.

Additionally, there is provided a method for treating unstable diabetes,the method comprising the step of administering to a subject in need ofsuch treatment a pharmaceutical composition described herein, therebytreating unstable diabetes. Further is provided a method for reducing anelevated fasting blood glucose level, the method comprising the step ofadministering to a subject in need of such treatment a pharmaceuticalcomposition described herein, thereby reducing an elevated fasting bloodglucose. In certain preferred embodiments, the subject is a humansubject.

Unstable Diabetes

Physicians skilled in the art will appreciate that unstable diabetes,also known as glycemic lability, can be diagnosed by a number ofacceptable standard procedures. One such procedure appears in Ryan etal, 2004. In this procedure, subjects are asked to monitor their glucoselevels with a minimum of 2 capillary glucose readings a day. Patientsrecord all measured glucose values and details about hypoglycemicoccurrences over a 4-week period on sheets (FIG. 11A-B). On any occasionthat glucose is recorded as <3.0 mmol/l, the subject is asked todescribe the details of the event on the questionnaire (FIG. 12),including which symptoms occur and whether outside help from a thirdparty is obtained to either recognize or treat the hypoglycemicreaction. A reaction is considered severe if the individual had lostcontrol of the situation and required outside help to treat thehypoglycemic event. Other such methods involve calculation of the MAGEindex (Service et al 1970) or the “M value” (Schlichtkrull et al) orutilize continuous glucose monitoring systems (Kessler et al). Unstablediabetes is typically associated with elevated fasting blood glucoseand/or hypoglycemic episodes.

Methods of Isolating Protease Inhibitors

In yet another aspect is provided a method for purifying BBI from asoybean product, e.g. soy flour, comprising the steps of: a) obtaining aliquid mixture comprising an extract from the soybean product, whereinthe liquid mixture is a pH-buffered aqueous solution having a pH ofbetween 5.5-7.5 inclusive, with a salt concentration of less than 50 mM;and b). subjecting the solution to size-exclusion chromatography,utilizing a stationary phase that comprises a positively charged resinand a polysaccharide polymer material, wherein the size-exclusionchromatography comprises a discontinuous step salt gradient, wherein thesalt concentration of the solution used in the first step is less than40 mM, preferably less than 20 mM, and the salt concentration of asolution used in a subsequent step (typically but not necessarily thesecond step) is between 40-85 mM, more preferably 50-80 mM, morepreferably 60-80 mM, more preferably 65-75 mM, most preferably 70 mM.

Alternatively or in addition, the above method further comprises theprevious step of extracting soy flour at buffer with a pH of 4.5, in thepresence of 80-120 mM NaCl, more preferably 100 mM NaCl, and in someembodiments subsequently adjusting the pH to 4.5, followed,alternatively or in addition, by clarification in a filter press.Alternatively or in addition, the method further comprises the previousstep (before the aforementioned step of obtaining a liquid mixturehaving a pH of between 5.5-7.5, but subsequent to extracting the soyflour) of precipitating the initial liquid preparation of the soy flourusing 30-40% saturation ammonium sulfate (AS), more preferably 35%saturation, in some embodiments under cooling, for example at atemperature of 12-17° C. In further embodiments, the precipitate is thencollected by centrifugation, for example in a continuous tubularcentrifuge. In still other embodiments, the AS pellet is extracted, incertain embodiments in a phosphate buffer of pH 7.0-8.0, then optionallyclarified by centrifugation. In various other embodiments, the resultingsupernatant is dialyzed against water, or alternatively 10 mM sodiumphosphate, pH 6.0-7.0 buffer, clarified by centrifugation, and/orlyophilized.

In more specific embodiments, the above method further comprises thesteps of isolating KTI by washing tire column with a buffer of 90-120mM, more preferably 100 mM, and eluting KTI with a buffer of over 150mM, preferably 180 mM. Alternatively or in addition, the method furthercomprises the step of removing microorganisms through filtration (anon-limiting example is a 0.45/0.2 μm filter). Alternatively or inaddition, the method further comprises the step of filtration though a20,000-40,000 MWCO filter, preferably 30,000 MWCO, and keeping thepermeate. This step is preferably done in the presence of a 0.15-0.5 Msalt concentration, more preferably 0.18 M. Alternatively or inaddition, the method further comprises the step of concentrating theresulting solution with a 5000 MWCO filter and keeping the retentate.Alternatively or in addition, the method further comprises the step ofdiafiltering the resulting solution with a 10,000 MWCO filter andkeeping the retentate, for example in sodium phosphate buffer having apH of 7.0-8.0. Alternatively or in addition, the method furthercomprises the step of lyophilizing the final liquid product.

In still another aspect is provided a method for purifying KTI3 (KunitzTrypsin Inhibitor 3) from a soybean product, comprising the steps of:a), obtaining a liquid mixture comprising an extract from the soybeanproduct, wherein the liquid mixture is a pH-buffered aqueous solutionhaving a pH of between 6-7 inclusive, with a salt concentration of lessthan 50 mM; and b) subjecting the solution to size-exclusionchromatography, utilizing a stationary phase that comprises a positivelycharged resin and a polysaccharide polymer material, wherein thesize-exclusion chromatography comprises a discontinuous step saltgradient, wherein the salt concentration of the solution used in a washstep (either the first step or a subsequent step) is more than 80 mM,and the salt concentration of a subsequent step (wherein foe KTI3 iseluted) is between 140-250 mM inclusive, preferably between 160-220 mMinclusive, more preferably between 170-200 mM inclusive, more preferably180 mM.

Alternatively or in addition, the above method further comprises foeprevious step of extracting soy flour at buffer with a pH of 4.5, in foepresence of 80-120 mM NaCl, more preferably 100 mM NaCl. Alternativelyor in addition, the method further comprises foe previous step ofprecipitating the initial liquid preparation of the soy flour using30-40% saturation ammonium sulfate, more preferably 35% saturation.Alternatively or in addition, the method further comprises the step ofremoving microorganisms through filtration (a non-limiting example is a0.45/0.2 μm filter). Alternatively or in addition, the method furthercomprises the step of concentrating the resulting solution with a 5000MWCO filter and keeping foe retentate. Alternatively or in addition, themethod further comprises the step of diafiltering the resulting solutionwith a 10,000 MWCO filter and keeping the retentate. Alternatively or inaddition, the method further comprises the step of lyophilizing foefinal liquid product.

The “liquid mixture” referred to above may in certain embodiments be asolution, a suspension, or a combination thereof. In preferredembodiments, its salt concentration is less than 40 mM, more preferablyless than 30 mM, more preferably less than 20 mM, more preferably lessthan 10 mM. Most preferably, no salt has been added. In otherembodiments, foe pH of foe liquid mixture is between 6-7 inclusive.

The positively charged resin used in the above methods may be in someembodiments a diethylaminoethyl (DEAE) tertiary amine functional group.Alternatively or in addition, the polysaccharide polymer material iscrosslinked through lysine side chains. One non-limiting example of sucha material is Sepharose™, a crosslinked, beaded-form of a polysaccharidepolymer material extracted from seaweed.

In certain embodiments, the described method of isolating proteaseinhibitors does not utilize a PEG-containing reagent.

Methods of Producing Pharmaceutical Compositions

Also provided herein are methods of producing pharmaceuticalcompositions. In certain embodiments, the method comprises the steps ofoptionally combining molten Gelucire (for example Gelucire 44/14) withfish oil, cooling the mixture, then adding, in powder form, EDTA, SBTI,aprotinin, and a therapeutic protein or peptide, for example insulin, orin other embodiments, exenatide, although those skilled in the art willappreciate in light of the present findings that other therapeuticproteins or peptides may be used as well. In other embodiments, themethod comprises the steps of optionally combining molten Gelucire (forexample Gelucire 44/14) with fish oil, cooling the mixture of, thenadding, in powder form, EDTA, BBI, and a therapeutic protein or peptide.In certain embodiments, the powder components are added in the listedorder. In other embodiments, the resulting mixture is optionally mutedand/or homogenized.

Wherever alternatives for single features such as purity of BBI, purityof KTI, nature of protein or nature of liquid component etc. are laidout herein as “embodiments”, it is to be understood that suchalternatives may be combined freely to form discrete embodiments of theentire formulation provided herein.

“Consisting essentially of”, as used herein, limits the scope of theinvention to the specified materials or steps and those that do notmaterially affect the basic and novel characteristics of the claimedinvention.

With respect to the jurisdictions allowing it, all patents, patentapplications, and publications mentioned herein, both supra and infra,are incorporated herein by reference.

EXPERIMENTAL DETAILS SECTION Example 1: Small-Scale Production ofImproved KTI and BBI Formulations Overview

Previous protocols for producing SBTI from soybean flour generated crudeprotease inhibitors at a yield of 1%. The following experiments wereperformed to develop a production method of separate bulk materials ofKTI (Kunitz Trypsin Inhibitor) with a Trypsin inhibitor (TI) activityand BBI (Bowman-Birk inhibitor) with a Trypsin/Chymotrypsin inhibitor(CTI) activity, where each product is prepared under its ownspecifications to high levels of enzymatic activity, the yield isimproved, and high molecular weight (MW)-contaminants are minimized.

The production process involves two major phases:

Part 1: Production of SBTI Intermediate from soy flour. Crude SBTI isprecipitated from the supernatant with ammonium sulfate (AS) andcollected by centrifugation. The protein is extracted and dialyzed toremove excess AS. The dialyzed extract is lyophilized, analyzed andtermed the “SBTI intermediate” (FIG. 1A).Part 2: Downstream purification of SBTI intermediate to produce purifiedBBI and KTI. The SBTI intermediate is loaded on a DEAE-Sepharose™ fastflow column. KTI and BBI are separately eluted by salt step gradients.The BBI is further purified to reduce the high-MW contaminants byfiltration through a 30,000 molecular weight cut-off (MWCO) filter. Thefinal formulation is prepared in phosphate buffer before lyophilization(FIG. 1B).

Various parameters of the processes shown in FIGS. 1(A) and (B) werealtered, including: the type of soy flour used as a starting material,the pH and salt level of the extraction buffer, the percentages of ASused for precipitation, the pH and salt level of the buffer used forcolumn loading, and the elution conditions (continuous salt gradient vs.step gradient).

7B soy flour (de-fatted and minimally heat processed soy flour, producedby Archer Daniels Midland Company Decatur, Ill.) was used. The preferredpH for extraction was 4.5, which yielded a much lower level ofnon-relevant proteins, while keeping the level of KTI and BBI relativelyhigh. A continuous material precipitation was observed in the absence ofsalt; the addition of salt to the extraction buffer improved the processconsiderably by preventing the precipitation. Use of 35% AS for proteinprecipitation resulted in a much lower level of high-MW proteins, whilethe level of KTI and BBI was minimally affected. Use of pH-6.5 columnloading buffer reduced the amount of non-relevant proteins bound to theDEAE-Sepharose column. Elution in salt gradient steps resulted in abetter separation of BBI from KTI and other non-relevant proteins. TheBBI was efficiently purified by filtering through a 30,000 MWCO filterin the presence of 0.5M NaCl.

Example 2: Larger-Scale Improved SBTI Production

Three larger-scale experiments using 25 kilograms (kg) soy flour wereperformed. Extract collection was performed with the use of a disk stackcentrifuge (Westfalia; first two experiments) or with a filter press(third experiment). 50% of the material was lost with the Westfaliacentrifuge, while no loss of extract was observed with the filter press.The protocol and the analytic results of the third experiment are shownin Tables 1-2, respectively.

TABLE 1 Large-scale SBTI Intermediate preparation Process Step ResultsFlour extraction: Mix 25 Kg 7 B flour with 250 L extraction buffer,containing 25 mM sodium phosphate and 100 mM NaCl, at pH 4.5, for onehour at room temperature, using an overhead stirrer. Adjust pH to 4.5with concentrated HCl. Clarification: add 7.5 kg. Hyflo and filter usingClear yellowish solution filter press. Wash filter press with 2 × 50 Lextraction buffer. Ammonium sulfate precipitation: 209 grams (gr)/Lfinal concentration (=35% percent saturation). Mix 2 hours at RT,incubate overnight at 12-17° C. To 490-540 L, with a cooling coil,gradually add the AS. Precipitation of AS pellet: Collect AS 1.4 Kg offirm brownish pellet precipitate by centrifugation in a continuoustubular centrifuge (Alfa Laval) at 1.8-2.2 L/min under suction.Extraction of AS pellet: Mix pellet with 12 L of 4 cycles of extractionwere required to 10 mM sodium phosphate, pH 7.6 buffer for 30 extractall the brownish solution from the min and centrifuge in a Sorvallcentrifuge, pellet Total volume = 15 L keeping the supernatant. Repeatthis step twice with 8 L and 4 L of buffer. Dialysis: Pool supernatantsat and dialyze in Beige, very hazy solution. Conductivity at 3,500 MWCObags against a total of 1 L water, the end of dialysis was 800 μSi for40-48 hours at 4-17° C. Clarification: by Sorvall centrifuge, 15 minutesLight brown clear suspension at 4800 RPM. Lyophilization: for 48-72hours Light brown powder, 444 gr. Final product* *Calculated value basedon dialysis of 2.1 liter (L) aliquot.

TABLE 2 Analytical Results of SBTI intermediated TEST RESULTS AppearanceBrown solid Protein percentage by BCA ≥102% Trypsin inhibitor activity 1mg of protein inhibits 1.34 mg of Trypsin having an activity of approx.10,000 BAEE units per mg. of protein Chymotrypsin inhibitor 1 mg shouldinhibit 0.98 mg of activity Chymotrypsin having an activity of approx.40 BTEE units per mg. of protein

Next, the SBTI intermediate was subjected to the downstream purificationprocess shown in Table 3. The analytical results of the obtained KIT andBBI are shown in Tables 4-5, respectively.

TABLE 3 Large-scale downstream preparation of purified BBI and KTI. StepResult Solubilize SBTI Intermediate: 20 mg/ml. in 1250 Clear brownishsolution, 1250 ml ml. of buffer A: 10 mM sodium phosphate, pH 6.5 DEAESepharose column chromatography: Conductivity of charge 3.09 mS/cm pH ofcharge Adjust to pH 6.5 Resin amount 840 ml Column size (diameter ×height) 10 cm × 10.7 cm Column volume (CV) 840 mL Flowrate 28 mL/minVolume of charge 1250 mL Volume of wash 8 L Elution with buffer Acontaining salt as follows: Collect fractions of 0.3 L 5 L (6 × CV) of 0to 0.14M NaCl (gradient) 5 L (6 × CV) of 0.14M NaCl (step) 5 L (6 × CV)of 0.14 to 0.18M NaCl (gradient) 5 L (6 × CV) of 0.18M NaCl (step) 3 Lof 0.4M NaCl (step) Collecting BBI pool 3.1 L clear solution CollectingKTI pool 4.5 L clear solution Filtration of BBI pool (only 840 ml): AddNaCl to 830 mL clear light yellow solution. final concentration of 0.5M.Filter through TFF Recovery by OD₂₈₀ = 61% 30,000 MWCO and collectpermeate Dialysis of KTI pool and filtered BBI pool in 1000 Clearsolutions. Conductivity of both MWCO bags against water for 2 days poolsis <100 uSi Formulation: Add 0.5M sodium phosphate, pH 7.6 to theprotein suspension so that the protein will be 90% in solidLyophilization: in Lyoguard trays for 48 hours *White powder of bothproteins: 10.2 gr of KTI and 2.25 gr of BBI

TABLE 4 Analytical results of purified KTI. TEST ANALYTICAL RESULTAppearance White Protein by BCA 99% Trypsin inhibitor activity 1 mginhibits 1.38 mg of Trypsin with activity of approx. 10,000 BAEE unitsper mg of protein Purity by SDS-PAGE and 100% imager Quantitation Highmolecular weight contaminants: <0.1%.

TABLE 5 Analytical results of purified BBI. TEST ANALYTICAL RESULTAppearance White Protein by BCA 99% Chymotrypsin inhibitor 1 mg inhibits1.49 mg. of activity Chymotrypsin with activity of approx. 40 BTEE unitsper mg of protein. Purity by SDS-PAGE and 89% BBI imager quantitationHigh molecular weight contaminants: <0.1%.

A clear separation of the KTI and BBI fractions was achieved (FIG. 2).The elution was performed with NaCl gradient from 0 to 0.14 M, from 0.14to 0.18 M and from 0.18 to 0.4 M. Conductivity and OD₂₈₀ were determinedfor each fraction. The pool of BBI was collected from fractions 18-28.The pool of KTI was collected from fractions 42-57.

The products of each pool were analyzed by SDS-PAGE. A high-purityproduct was obtained in each case (FIG. 3).

Thus, significant improvements in the synthetic method and the resultingproduct were achieved.

TABLE 6 A summary of the characteristics of the different preparations.Previous Step procedure Small-scale prep Large-scale prep SBTIintermediate preparation Starting material (flour) 1,013 Kg 0.2 Kg 25 KgClarification of flour — 7.84 gr protein 1,862 gr (7.5%) extraction(3.92%) SBTI intermediate 10 kg 3.5 gr 444 gr Protein yield 0.98% 1.75%1.78% Downstream SBTI purification Protein Loaded on 2,000 gr 3.5 gr 30gr column Final product 0.449 2.31 gr BBI (BBI + KTI) 10.35 gr KTIPurification yield 62% BBI 13-17% of a 7.4% of BBI and and 61% formixture of both 34% of KTI KTI proteins Degree of BBI + +++ +++purification BBI Activity 1.2 0.03-0.3  1.49 KTI activity 1.29 1.5-1.81.34

Example 3: Alternative Downstream BBI and KTI Purification ProtocolDescription of Changes

Several changes were made to the downstream purification protocol:

1. Addition of Thimerosol to the Pools Collected from the Columns.

The BBI lots prepared without thimerosal were found to contain a hightotal aerobic microbial count (CFU/G) that was above the bio-burdenspecification of ≤100 CFU/G. The total aerobic microbial count results(CFU/G) were: KTI, Lot 1: 0; BBI, Lot 2: 15.4; BBI, Lot 3: 61.6; BBI,Lot 4: 15.4. Therefore, thimerosal was added to the pools that werecollected from the column to a 0.01% final concentration. All thesubsequent lots were found to be within the bio-burden specification.The total yeast and mold counts were zero for all lots.

2. Improved Salt Gradients.

In order to better separate the BBI from KTI, a stepwise salt gradientwas performed, using pre-prepared buffers with known conductivity.

3. Improved Filtration Process

The 30,000 MWCO filtration process was adapted to use the KVICK™ holderoutside the system instead of the Uniflux10™ system. The use of theKVICK holder enabled cycling of the material through the filters andhandling large volumes at a time. The filtration was performed in acontained fashion, to the extent possible.

This change was found to minimize loss of protein and BBI activity.Prior to the change, 40-70 gram of final product with a BBI activity of0.4-0.6/mg was obtained per purification lot. Afterwards, 85-135 gram offinal product with a BBI activity of 0.8-1.4/mg was obtained. Also, thesolution used for the ultrafiltration was changed from 0.5 M NaCl to0.18 M NaCl.

Detailed Protocol

The downstream purification was performed in a cGMP facility, startingwith 800 gr and 350 gr batches of SBTI intermediate. The intermediatewas suspended with 40 L buffer A and loaded on the DEAE column (37L=CV). The quality control (QC) results for the loaded material were31.47 mgP/ml; KTI: 0.97; BBI: 0.09. The column was washed sequentiallywith:

1) 4×CV buffer A (10 mM sodium phosphate pH 6.5, 1.5 mSi (milliSiemensper meter);2) 2.1×CV buffer A1 (10 mM sodium phosphate pH 6.5+75 mM NaCl, 9.9 mSi);3) 3×CV buffer A2 (10 mM sodium phosphate pH 6.5+100 mM NaCl, 12 mSi);and4) 4×CV buffer A3 (10 mM sodium phosphate pH 6.5+180 mM NaCl, 21 mSi).

The BBI pool consisted of fractions F5-F11 and had a volume of 130 L and1.46 mgP/ml. The KTI pool consisted of fractions F15-F18 and had avolume of 85 L. Thimerosal was added to both pools to finalconcentration of 0.01%. Stock NaCl solution was added to the BBI pool toa final concentration of 0.5 M NaCl.

Next, the BBI pool was subjected to filtration, using the Kvick holder,with five 30,000 MWCO cassettes. The permeate was retained. The KTI poolwas concentrated 5-fold by the Uniflux system with 3 cassettes of 10,000MWCO to a final volume of 17.5 L. The concentrated pool was diafiltratedagainst water by the Uniflux10 system with 3 10,000 MWCO cassettes.

To test the efficacy of the above protocol, small-scale columnchromatography was performed, and the different fractions were tested,yielding the improved results shown in FIGS. 4-5, respectively. Theseresults were found to be reproducible over several runs.

A similar protocol was repeated to purify BBI only, except that 3.5×buffer A having 12.4 mSi was utilized, and the buffer A3 elution was notperformed. Additionally, after ultrafiltrating the BBI pool (135 L) with2 KVICK holders, using 30,000 MWCO filters (2×0.55 m²), the permeate wasthen concentrated 4.5 times, using the same system but with 5,000 MWCOfilters. The concentrated retentate was diafiltrated against water inthe Uniflux system. Samples of each purification step and the finalproduct were tested by SOS-PAGE, according to the protocol below;results are shown in FIGS. 6 and 7, respectively.

The SDS-PAGE utilized ready-to-use gels (PhastGels™, 20%, from AmershamPharmacia) run in a Phast™ system (Pharmacia). The staining solution was50% methanol, 10% acetic acid, and 0.05% Brilliant Blue. The de-stainingsolution was 30% methanol, 10% acetic acid.

A sample from each step was analyzed for protein concentration and BBIactivity. The results are summarized in Table 7. For the final product,the quantified imaging showed $4.4%-84.9% for the BBI band.

TABLE 7 Yield of protein and activity during the BBI purification steps.BBI activity (mg ChymoTrypsin Total inhibited/1 mgP- Total BBI BCAVolume protein ChymoTrypsin activity, units Step (mgP/ml) (L) (gr) (%)Inhibitor) (%) Column Load 40 1,100 BBI pool 2.1 135 283 (25.7%) 0.5141,500 BBI permeate 0.84 173 145 (13%) 0.8 116,000 (82%) of 30 Kda.Retentate of 30 41.6 2.5 104 (9.45%) 0.13 13,500 (9.5%) KDa Retentate of5 3.84 36 138.24 (12.5%) 0.8 110,600 (78%) KDa after 4.5 ×concentration. Permeate of 5 0.12 120 14.4 0.13 1,872 KDa Retentate of10 4.03 36 145 (13%) 0.7 101,500 (71%) Kda after diafltration Finalafter 1.07 136 gr 136 gr 0.8/mg 108,800 (77%) lyophilization mgP/mgSsolid solid solid* “as-is” (12.3%) *The material contains 10% water,therefore the result of activity is 0.9 on the basis of dry weight.

A flow chart of the improved protocol is shown in FIG. 8.

Prior to the improvements described in Examples 1-3, large-scale SBTIpurifications yielded 40-70 gram of final product per purification lot,with a BBI activity of 0.4-0.6/mg (while somewhat higher activity wasobtained in smaller-scale preparations, this could not be successfullyscaled-up). The changes enabled a yield of 85-135 gram of final productwith a BBI activity of 0.8-1.4/mg.

Example 4: Downstream BBI and KTI Purification Protocol UsingMicrofiltration

The protocol in this Example was performed without thimerosal. Startingwith the SBTI intermediate, DEAE column chromatography was performedsimilarly to the last Example but with slightly different parameters, asfollows:

3×CV buffer A (10 mM sodium phosphate pH 6.5, 1.5 mSi)2.1×CV buffer A1 (10 mM sodium phosphate pH 6.5+70 mM NaCl, 9.9 mSi)3.5×CV buffer A2 (10 mM sodium phosphate pH 6.5+100 mM NaCl, 12.4 mSi)3.5×CV buffer A3 (10 mM sodium phosphate pH 6.5+180 mM NaCl, 21 mSi)

Results are shown in FIG. 9.

BBI Pool

The BBI pool, 130 L, was collected and filtered through a Sartobran™0.45/0.2 μm filter (0.6 m²).

KTI Pool

The KTI, 95 L, was collected and filtered through a Sartobran™ 0.45/0.2μm filter (0.6 m²) and stored at −20° C. Final volume=20 L.

BBI Purification 30,000 MWCO Filtration

The BBI pool was adjusted to 0.18 M NaCl concentration and subjected tofiltration using 2×Kvick™ holders, each with 5 cassettes of 30,000 MWCO.The permeate, after four washes, was collected, 195 L total

Concentration

The permeate of the 30,000 MWCO was concentrated 5 times through 2 KVICKholders, each with five 5,000 MWCO cassettes in each. Final volume=34 L.

Diafiltration

The concentrated material was diafiltrated against purified water usinga Uniflux10 system with 5×10,000 MWCO filters to a conductivity of lessthan 1 mSi/cm.

Formulation buffer (0.5 M sodium phosphate buffer, pH 7.6) was added,and the sample was passed through a 0.45/0.2 μm filter directly intoGore® Lyoguard® Freeze-Drying Trays.

Lyophilization

After lyophilization, 95 gr. of dry material was obtained and collectedinto glass amber bottles. All bottles were QC sampled: The water contentwas found to be 8% by the Karl Fischer method.

Two duplicate samples were tested by SDS-PAGE, and the gel wasquantified using an imager. The BBI in both lanes was at 93.5-93.8%purity (FIG. 10).

Samples from each BBI purification step were tested for proteinconcentration, BBI activity and bio-burden. Table 8 summarizes the BBIquantity and quality along the purification process:

TABLE 8 BBI purification steps, quantity,characteristics, andbio-burden. BBI (mg ChymoTrypsin Total inhibited/1 mgP- Bio-burden BCAVolume protein ChymoTrypsin Total BBI (TAMC) Step (mgP/ml) (L) (gr)inhibitor) activity (CFU/ml) Column Load NA 46 1,100 NA NA TNTC BBI pool1.4 130 182 0.6 109,000 TNTC BBI pool after 1.4 130 182 0.6 109,000 1microfiltration 0.45/0.2 BBI permeate, after 30 0.7 195 136.5 0.8109,000 10  Kda UF and additional washes Retentate of 5 KDa 3.6 34 122.50.9 110,250 148  after 5x concentration Retentate of 10 Kda 3.1 34 1051.0 105,000 TNTC after diafiltration BBI After formulation 3.0 34 1020.8 81,600 0 and microfiltration 0.45/0.2 Final after — — 95 gr solid0.9 85,500 0 lyophilization TNTC = too numerous to count

KTI Purification Concentration of KTI

The microfiltrated KTI pool was taken out of cold storage andconcentrated using 2×KVICK holders with 5 5KDa MWCO filters each. Finalvolume=17 L

Diafiltration of KTI

The concentrated material was diafiltrated against water by Uniflux with5×10,000 MWCO filters. Final volume=20 L

Formulation buffer was added, and the sample was passed through a0.45/0.2 μm filter directly into Gore lyophilization trays.

Lyophilization

The lyophilized product was removed and transferred to 4 amber glassbottles.

Samples from each KTI purification step were tested for proteinconcentration, KTI activity and bio-burden. Table 9 summarizes the KTIquantity and quality along the purification process:

TABLE 9 KTI purification steps, quantity, characteristics, andbio-burden. KTI (mg Trypsin Total inhibited/1 mgP- Total Bio-burden BCAVolume protein Trypsin KIT (TAMC) Step (mgP/ml) (L) (gr) Inhibitor)activity (CFU/ml) Column Load NA 46 1,100 NA NA TNTC KTI pool 4.5 95427.5 1.5 641,250 TNTC KTI pool after 4.3 95 408.5 1.5 612,750 TNTCmicrofiltration 0.45/0.2 Retentate of 5 24.7 17 419.9 1.3 545,870 4.2 ×10⁷/m1 KDa after 5x concentration Retentate of 10 18 20 360 1.5 540,0003.4 × 10⁷/ml Kda after diafiltration KTI After 18 20 360 1.4 504,000 0.0formulation and microfiltration 0.45/0.2 Final after — — 300 gr Not yetNot yet lyophilization solid

Conclusions

1. The SBTI intermediate has a relatively high bio-burden, which can beremoved by the second microfiltration.2. The final products following microfiltration are within the requiredspecifications.3. The use of microfiltration has no significant effect on the yield orspecific activity of BBI and KTI.

Example 5: Identification of Effective Emulsifiers for HomogenousInsulin/Fish Oil Preparations

Previous formulations of insulin in fish oil were found to slowlyprecipitate; they were thus unsuitable for large-scale pharmaceuticaldosage form preparation. New formulations containing 3.375 g. SBTI per22.5 g. of fish oil and containing the following emulsifiers weretested: lecithin (trial sequence 1), Polysorbate 80 (Tween-80) (sequence2), or Gelucire 44/14 (sequence 3), alone or in combination with eachother or glycerol monostearate (GMS) (FIG. 11). Subsequently, the mostpromising formulations (indicated with an asterisk) were produced againby melting the Gelucire (which was a waxy solid as ambient temperature),then adding it to the fish oil. After cooling this mixture, the solidcomponents were added in powder form in the following order: EDTA, SBTI,aprotinin, and insulin; and the resulting liquid was mixed andhomogenized on a roller mill.

Example 6: In Vivo Animal Testing of Various Emulsifier Formulations

Materials and Experimental Methods

Formulations

The formulations that were tested are shown below in Table 10. Thepercentages of emulsifiers given are weight/weight with respect toweight of the liquids present.

TABLE 10 Formulations used in this Example. Formulation name EmulsifiersOther ingredients Experiment 6A GMS 2% GMS 2% only 75 mg SBTI, 150 mgEDTA, 24 mg aprotinin, 8 mg human insulin, 0.5-0.7 ml of fish oil 2%-2%2% GMS, 2% lecithin Same as above. 2%-10% 2% GMS, 10% lecithin Same asabove. 10% lec. 10% lecithin only Same as above. Experiment 6B A 5%lecithin, 2% GMS Same as above. B 3% lecithin, 12% Gelucire 44/14 Sameas above. C 6% lecithin, 12% Gelucire 44/14 Same as above. D 5%Tween-80, 12% Gelucire 44/14 Same as above. E 10% Tween-80, 12% Gelucire44/14 Same as above. F 12% Gelucire 44/14 only Same as above.

Husbandry

Animals: Only healthy pigs, as certified by a clinical veterinarian,were used for the study. Housing: Solitary when with CVC and grouped atother times. Bedding: Concrete+woodchips. Illumination: 12-12 h lightcycle. Temperature: 19-25° C.

Identification

Each animal was uniquely identified via ear tags.

Experimental Design

Animals were deprived of food 24-36 hours prior to testing and duringthe ensuing monitoring period. Access to water was ad libitum.

Animals were anesthetized with 20 mg/kg ketamine+2 mg/kg xylazine.Fasting and anesthetized pigs were positioned on their left side beforeliquid formulations were administered under endoscopic guidance,directly to the duodenum. After injection of the formulation, 1 ml. fishoil was injected, followed by 10 ml. air, to flush the apparatus,thereby ensuring administration of all materials. Pigs were thenreturned to their pens to allow for full recovery from the anesthetictreatment, which required 10-15 min. Blood samples (0.5 ml. of whichwere tested) were periodically drawn from the central line catheter(CVC) over the ensuing 240-min monitoring period. Blood glucoseconcentrations were determined from each sample, at each lime point.Piglets were intravenously treated with gentamycin (100 mg/10 kg) afterevery experiment day to avoid infection. In cases where glucoseconcentrations dropped below 30 mg/dL, piglets were served commercialpig chow, and glucose concentrations were monitored for an additional 30minutes thereafter.

A washout period of at least 2 days was enforced between test days.

Results

10 insulin-fish oil formulations with different emulsifiers were testedfor in-vivo activity on blood glucose levels in 2 separate experiments.Results are shown in Table 11.

TABLE 11 Results of Experiment 6A. The three numbers in each boxindicate baseline value, lowest value, and end (20 mg/dL). “Low”indicates a value of less than 20 mg/dL. Pig 5 Formulation Pig 1 Pig2Pig3 Pig4 (stoma) Score* GMS 2% 78 → 48 → 72 → low → 63 → 23 → 67 → 32 →55 → 21 → 3 65 low 28 60 58 2%-2% 82 → Low → 78 → 45 → 85 → 30 → 78 → 23→ 2 65 76 68 61 2%-10% 76 → Low → 76 → low → 76 → low → 74 → low → 4 7721 low 70 10% lec. 51 → 63 → 50 → low → 57 → low → 61 → low → 5 71 lowlow low *see FIG. 11 legend.

The results of Experiment 6B, depicted in FIG. 12(A)-1 to FIG. 12(A)-4;FIG. (12B)-1 and 12(B)-2, show that while all the formulations wereefficacious, formulations E and F from Table 10 induced the mostuniformly sharp drops in glucose levels.

Example 7: Clinical Testing of Oral Protein Formulations ContainingImproved SBTI

A study was performed to determine the safety, pharmacokinetics, andpharmacodynamics of the described oral insulin formulations in healthyvolunteers. Subjects received a single dose of one of the following oralinsulin tablet formulations on separate visits. Each dose was followedby a 72-hour washout period. Doses were administered in the morningafter an 8-hour overnight fast.

Treatment Groups

-   -   1. 1 capsule of 8-mg Insulin Formulation: 8 mg Insulin, 150 mg        EDTA, 75 mg total of a mixture of purified BBI and purified KTI,        150000U Aprotinin, and 12% Gelucire 44/14.    -   2. 1 capsule of 16-mg Insulin Formulation: 16 mg Insulin, 150 mg        EDTA, 75 mg total of a mixture of purified BBI and purified KTI,        150000U recombinant Aprotinin and 12% Gelucire 44/14.    -   3. 2 capsules of 8-mg Insulin Formulation.

Screening Phase:

The following evaluations were performed after the subject signedinformed consent:

-   -   Medical history    -   Physical examination    -   Medication history    -   ECG    -   Vital signs (blood pressure, heart rate). Vital signs were        measured in the sitting position after at least 5 minutes of        rest.    -   Clinical laboratory evaluations (chemistry, hematology)

Treatment Phase

Subjects entered the clinic on the morning of dosing after an 8-hourovernight fast.

Prior to study drug administration:

-   -   An indwelling catheter was inserted for blood sample collection.    -   A glucose test (one drop) was performed IS minutes (min) prior        to study drug administration    -   Vital signs were recorded 20 min prior to study drug        administration. Vital signs were measured in the sitting        position after at least 5 min of rest.    -   Blood samples for insulin, plasma glucose and c-peptide analysis        were collected 15 min prior to study drug administration.

Protocol for Drug Administration:

-   -   The experimental drug was administered orally with at least 300        mL of water. Subjects remained in an upright or sitting position        for at least one hour after taking study medication.    -   Vital signs (blood pressure, heart rate) were recorded at        approximately 2 and 5 hours (hr) post study drug administration.        Vital signs were measured in the sitting position after at least        5 min of rest.    -   Blood samples for insulin, plasma glucose, and c-peptide        analysis were collected every 20 min during the first hr. and        then every 15 min up to 5.0 hr post study drug administration.

After completing the evaluations for each period, the subjects were fedand discharged, then asked to return for the next period after a minimumof 72-hour washout period.

End of Study/Early Discontinuation:

Prior to discharge from the research unit, subjects underwent thefollowing end-of-study evaluations:

-   -   Vital signs were measured in the sitting position after at least        5 min of rest.    -   Clinical laboratory evaluations (hematology, chemistry)

Subjects that discontinued early completed the end-of-study evaluationsat the time of discontinuation.

Results

The various doses of insulin were well tolerated, with no adverse eventsreported or observed following any of the treatments. Responses weredemonstrated in 7/10 subjects, with maximal blood glucose responses forall treatments observed following a lag of ≥60 min from administration.Significantly lower mean blood glucose C_(min) was observed followingthe 8+8 mg (47.9±11.3 mg/dL, p=0.006) and 16 mg (57.3±8.4 mg/dL,p=0.001) treatments, when compared to the 8-mg group (64.6±62 mg/dL).Significant reductions in glucose area under the curve (AUC) values wereobserved following both 8+8 mg and 16 mg treatments (13.2% and 8.1%,respectively), when compared to 8 mg (p=0.003 and 0.008, respectively)(FIG. 15 and Table below). Such differences were not seen withformulations containing prior art SBTI preparations.

Summary of Area-Under-the-Curve (AUC) Data Following InsulinAdministration

Treatment Avg AUC (mg/dL*min) St-dev (mg/dL*min) 8 22425 2153 16 206101428 8 + 8 19471 2126

Example 8: Animal Testing of Oral Protein Formulations ContainingImproved SBTI

Additional animal testing is performed on formulations comprising highlypurified SBTI and a protein drug (e.g. insulin or exenatide), in amanner similar to one or more of the protocols described herein. In someexperiments, liquid formulations are coated with a gelatin and/orenteric-coated capsule, which may be administered orally. In otherexperiments, liquid formulations are administered directly to theduodenum via a cannula or the like. In certain experiments, animals areallowed to eat prior to or following administration of the formulation,to simulate pre-prandial or post-prandial conditions.

In still other experiments, recombinant SBTI is used as described above.In other experiments, synthetic SBTI is used as described above.

Example 9: Testing of Oral Exenatide Formulations Containing ImprovedSBTI in Human Subjects

The following study is being performed to assess the safety,pharmacokinetics and pharmacodynamics of oral exenatide formulationscomprising purified BBI and KTI and optionally 12% Gelucire 44/14 inhealthy volunteers and in T2D subjects, with screening and treatmentperformed similarly to Example 7:

Stage I: Stage I consists of two segments: Segment 1 will assess thesafety, tolerability and the PK/PD of escalating doses of oral exenatidein healthy volunteers. In the first segment, the two lower doses ofexenatide (ISO and 300 μg) will be randomly administered to all healthy,fasting subjects. If deemed safe, further dose escalation (450 and 600μg exenatide) will be authorized in Segment 2. The highest tolerabledose will then be administered to all healthy patients 60 minutes beforea standard meal (Visit 5).Stage II: This stage will assess the T2DM patient response to escalatingdoses of exenatide when delivered 60 min before a standard meal. Placebocontrols may be included in the study, as may treatment with an activecontrol of Byetta (5 μg) subcutaneously delivered 30 min before astandard meal. In addition, all T2DM subjects will be treated with anoral insulin capsule containing 16 mg of insulin and with a combinationof oral insulin/oral exenatide, at two independent study visits, 60minutes before being served a standard meal.

Interpretation:

AUC of glucose reductions and insulin excursion will be calculated andcompared between the different treatments. The efficacy of theformulation will be shown by insulin excursions in the oralGLP-1-treated group that are greater than those in the non-GLP-1-treatedgroup, and/or smaller glucose excursions in the oral GLP-1-treatedgroup.

In still other experiments, recombinant BBI (optionally with KTI and/oraprotinin) is used as described above in place of purified BBI and KTI.In other experiments, synthetic BBI (optionally with KTI and/oraprotinin) is used as described above in place of purified BBI and KTI.

Example 10: Testing of Oral Insulin Formulations Containing ImprovedSBTI in Human Subjects

The following study is being performed to assess safety andpharmacodynamics of multiple bedtime doses of oral insulin formulationscomprising purified BBI and KTI and optionally 12% Gelucire 44/14, inadult patients with T2DM who are inadequately controlled with diet andmetformin, with screening and treatment performed similarly to Example7:

Primary Objectives: To evaluate the pharmacodynamic effects of theformulation on mean nighttime glucose levels and safety parameters(e.g., hypoglycemia), and to evaluate safety, including incidence ofhypoglycemia and cardiovascular events. Primary efficacy endpoints willbe determined by:

-   -   The effect of 24 mg insulin on weighted mean nighttime glucose        levels based on two nights of (continuous glucose monitoring        (CGM)) data*, determined by comparison of:    -   a) The mean percent change between baseline (run-in period) and        24 mg insulin treatment to    -   b) The mean percent change between baseline and placebo        treatment for the group receiving placebo.    -   “CGM data” as used herein is always based entirely on data        collected within the first six hours after oral medicinal        treatment.    -   The effect of 16 mg insulin on weighted mean nighttime glucose        levels based on two nights of CGM data determined by comparison        of:    -   a) The mean percent change between baseline (run-in period) and        16 mg insulin treatment to    -   b) The mean percent change between baseline and Week 4 of        placebo treatment for the group receiving placebo.        Secondary Objectives: To evaluate changes in baseline in fasting        blood glucose (FBG), morning fasting serum insulin, c-peptide,        triglycerides, and HbAlc. Secondary endpoints will be determined        by:    -   The effect of 24 mg insulin and/or 16 mg insulin on weighted        mean nighttime glucose levels will determined by comparison of        weighted mean nighttime glucose levels for insulin-treated        groups vs. the group receiving placebo.    -   The effect of 24 mg insulin and/or 16 mg insulin on changes from        baseline to the treatment phase in fasting morning blood        glucose, morning fasting serum insulin, morning fasting        c-peptide, HbAlc, and triglycerides.    -   The effect of 24 mg insulin and/or 16 mg insulin on changes from        baseline to the treatment phase in mean glucose assessed by CGM.

In still other experiments, recombinant BBI (optionally with KTI and/oraprotinin) is used as described above in place of purified BBI and KTI.In other experiments, synthetic BBI (optionally with KTI and/oraprotinin) is used as described above in place of purified BBI and KTI.

Example 11: Testing of Oral Insulin Formulations Containing ImprovedSBTI in Treatment of Unstable Diabetes

Subjects with unstable diabetes (for example, subject having a glycatedhemoglobin [HgA1c] level of 8-10%) are monitored for several days usinga blinded continuous glucose monitor (CGM) to establish a baseline.During several subsequent days, they are administered aninsulin-containing formulation described herein, optionally using priorart formulations as a control group, prior to meals. Blinded CGM isperformed to determine the efficacy of the formulations.

In still other experiments, recombinant BBI (optionally with KTI and/oraprotinin) is used as described above in place of purified BBI and KTI.In other experiments, synthetic BBI (optionally with KTI and/oraprotinin) is used as described above in place of purified BBI and KTI.

Testing of Oral Insulin formulations Containing Improved BBI in theAbsence of Other Protease Inhibitors

Additional studies are performed, similar to those described above, butusing BBI prepared as described herein, in the absence of KTI (e.g. BBIand aprotinin as the only protease inhibitors), and in otherexperiments, in the absence of any other protease inhibitors. Theability of the improved BBI alone to protect protein drugs in thedescribed formulations in the absence of other protease inhibitorsconfirms the superiority of BBI prepared as described herein.

In the claims, the word “comprise”, and variations thereof such as“comprises”, “comprising”, and the like indicate that the componentslisted are included, but not generally to the exclusion of othercomponents.

REFERENCES

-   Eldor R, Kidron M, Arbit E. Open-label study to assess the safety    and pharmacodynamics of five oral insulin formulations in healthy    subjects. Diabetes Obes Metab. March 2010A; 12(3):219-223.-   Eldor R, Kidron M, Greenberg-Shushlav Y, Arbit E. Novel    glucagon-like peptide-1 analog delivered orally reduces postprandial    glucose excursions in porcine and canine models. J Diabetes Sci    Technol. 2010B; 4(6):1516-1523.-   Kessler L, Passemard R, Oberholzer J et al: Reduction of blood    glucose variability in type 1 diabetic patients treated by    pancreatic islet transplantation. Diabetes Care 25:2256-2262, 2002.-   Kidron M, Dinh S, Menachem Y, et al. A novel per-oral insulin    formulation: proof of concept study in non-diabetic subjects. Diabet    Med. April 2004; 21 (4):354-357.-   Nissan A, Ziv E, Kidron M, et al. Intestinal absorption of low    molecular weight heparin in animals and human subjects. Haemostasis.    September-October 2000; 30(5):225-232.-   Ryan E A, Shandro T, Green K et al. Assessment of the severity of    hypoglycemia and glycemic lability in type 1 diabetic subjects    undergoing islet transplantation. Diabetes. 2004 April,    53(4):955-62.-   Schlichtkrull J, Munck O, Jersild M: The M-value, an index of    blood-sugar control in diabetics. Acta Med Scand. 177:95-102, 1965.-   Service F J, Molnar G D, Rosevear J W et al. Mean amplitude of    glycemic excursions, a measure of diabetic instability. Diabetes.    1970 September; 19(9):644-55.-   Siepmann F, Siepmann J et al, Blends of aqueous polymer dispersions    used for pellet coating: importance of the particle size. J Control    Release. 2005; 105(3): 226-39.-   Sprecher C A, Morgenstem K A, Mathewes S, Dahlen J R, Schrader S K,    Foster D C, Kisiel W. J Biol Chem. 1995 Dec. 15; 270(50):29854-61.-   Sun J., Rose J. B., Bird P. (1995) J. Biol. Chem. 270, 16089-16096.-   Ziv E, Kidron M, Raz I, et al. Oral administration of insulin in    solid form to nondiabetic and diabetic dogs. J Pharm Sci. June 1994;    83(6):792-794.

1.-33. (canceled)
 34. An oral pharmaceutical composition produced by aprocess comprising (a) subjecting soybean trypsin inhibitor (SBTI) tocolumn chromatography under conditions in which fractions containing theSBTI's Bowman-Birk Inhibitor (BBI) activity elute separately fromfractions containing the SBTI's Kunitz Trypsin Inhibitor (KTI) activity;(b) eluting and combining fractions from (a) that contain the BBIactivity; (c) filtering the combined fractions from (b) that contain theBBI activity under conditions that reduce the contaminants having amolecular weight of greater than 30 KDa to be less than 0.1% of the BBIpreparation, thus producing a purified BBI product characterized inthat: (i) contaminants having a molecular weight greater than 30 KDa areless than 0.1% of the preparation; and (ii) 1 mg of the purified BBIproduct has an activity of about 40 BTEE units per mg of protein; (d)eluting and combining the fractions from (a) that contain the KTIactivity and in which contaminants having a molecular weight greaterthan 30 KDa are less than 0.1% of the preparation thus producing apurified KTI product characterized in that: (i) contaminants having amolecular weight greater than 30 KDa are less than 0.1% of thepreparation; and (ii) 1 mg of the purified KTI product has an activityof about 10,000 BAEE units per mg of protein; (e) combining (i) anoil-based liquid formulation, wherein the oil comprises fish oil, (ii) atherapeutic protein having a molecular weight of up to 100 kilodalton,(iii) a chelator of divalent cations, (iv) the purified BBI product ofpart (c); and (v) the purified KTI product of part (d), such that theratio of anti-chymotrypsin activity to anti-trypsin activity present inthe pharmaceutical composition is between 1.5:1 and 1:1 inclusive,wherein the composition is water-free and comprises a coating thatresists degradation in the stomach.
 35. An oral pharmaceuticalcomposition comprising an oil-based liquid formulation, wherein the oilin the oil-based liquid formulation comprises fish oil, and wherein theoil-based liquid formulation further comprises: a therapeutic protein ofup to 100 kilodalton, a chelator of divalent cations, and a Bowman-BirkInhibitor (BBI), wherein the composition is water-free and comprises acoating that resists degradation in the stomach, wherein the BBI has aratio of anti-chymotrypsin activity to anti-trypsin activity present ofbetween 1.5:1 and 1:1 inclusive, and wherein the BBI has been producedby a process comprising: (a) subjecting soybean trypsin inhibitor (SBTI)to column chromatography under conditions in which fractions containingthe SBTI's Bowman-Birk Inhibitor (BBI) activity elute separately fromfractions containing the SBTI's Kunitz Trypsin Inhibitor (KTI) activity;(b) eluting and combining the fractions from (a) that contain the BBIactivity; and (c) filtering the combined fractions from (b) that containthe BBI activity under conditions that reduce contaminants having amolecular weight of greater than 30 KDa to be less than 0.1% of the BBIpreparation, thus producing a purified BBI product characterized inthat: (i) contaminants having a molecular weight greater than 30 KDa areless than 0.1% of the preparation; and (ii) 1 mg of the purified BBIproduct has an activity of about 40 BTEE units per mg of protein.
 36. Amethod for making a pharmaceutical composition, the method comprising(a) providing a preparation of Bowman-Birk Inhibitor (BBI), wherein theBBI has been produced by a process comprising (i) subjecting soybeantrypsin inhibitor (SBTI) to column chromatography under conditions inwhich fractions containing the SBTI's Bowman-Birk Inhibitor (BBI)activity elute separately from fractions containing the SBTI's KunitzTrypsin Inhibitor (KTI) activity; (ii) eluting and combining thefractions from (i) that contain the BBI activity (iii) filtering thecombined fractions from (ii) that contain the BBI activity underconditions that reduce contaminants having a molecular weight of greaterthan 30 KDa to be less than 0.1% of the BBI preparation, thus producinga purified BBI product characterized in that: contaminants having amolecular weight greater than 30 KDa are less than 0.1% of thepreparation; and 1 mg of the purified BBI product has an activity ofabout 40 BTEE units per mg of protein; and (b) mixing the purified BBIproduct of part (iii) and a therapeutic protein of up to 100 kilodaltonsinto an oil-based liquid formulation in which the oil comprises fishoil; wherein the preparation of BBI that is mixed with the therapeuticprotein is of a purity of at least 85% as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE); and wherein theratio of anti-chymotrypsin activity to anti-trypsin activity present inthe pharmaceutical composition is between 1.5:1 and 1:1 inclusive. 37.The method of claim 36, wherein the oil in the formulation is fish oil.38. A method for making an oral pharmaceutical composition comprising(a) subjecting soybean trypsin inhibitor (SBTI) to column chromatographyunder conditions in which fractions containing the SBTI's Bowman-BirkInhibitor (BBI) activity elute separately from fractions containing theSBTTs Kunitz Trypsin Inhibitor (KTI) activity; (b) eluting and combiningthe fractions from (a) that contain the BBI activity; (c) filtering thecombined fractions from (a) that contain the BBI activity underconditions that reduce contaminants having a molecular weight of greaterthan 30 KDa to be less than 0.1% of the BBI preparation, thus producinga purified BBI product characterized in that: (i) contaminants having amolecular weight greater than 30 KDa are less than 0.1% of thepreparation; and (ii) 1 mg of the purified BBI product has an activityof about 40 BTEE units per mg of protein; (d) eluting and combining thefractions from (a) that contain the KTI activity and in whichcontaminants having a molecular weight greater than 30 KDa are less than0.1% of the preparation thus producing a purified KTI productcharacterized in that: (i) contaminants having a molecular weightgreater than 30 KDa are less than 0.1% of the preparation; and (ii) 1 mgof the purified KTI product has an activity of about 10,000 BAEE unitsper mg of protein; (e) combining (i) an oil-based liquid formulation,wherein the oil comprises fish oil, (ii) a therapeutic protein having amolecular weight of up to 100 kilodalton, (iii) a chelator of divalentcations, (iv) the purified BBI product of part (b); and (v) the purifiedKTI product of part (c), such that the ratio of anti-chymotrypsinactivity to anti-trypsin activity present in the pharmaceuticalcomposition is between 1.5:1 and 1:1 inclusive.
 39. The method of claim38, wherein the oil in the formulation is fish oil.